JP2022509809A - Nucleic acid for inhibiting C3 expression in cells - Google Patents

Abstract

The present invention relates to nucleic acid products that interfere with or inhibit the expression of the complement component C3 gene. The nucleic acid is preferably a complement component C3-related disease, disorder or syndrome, particularly C3 glomerulocytosis (C3G), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic urinary syndrome (aHUS), For use as a treatment, prevention or reduction of risk of lupus nephritis, IgA nephropathy (IgA N), cryoaggregatosis (CAD), severe myasthenia (MG), and primary membranous nephropathy It is a thing.

Description

The present invention relates to nucleic acid products that interfere with or inhibit C3 complement component gene expression. The present invention further relates to such inhibition, such as for the treatment of diseases and disorders associated with deregulation and / or overactivation of the complement pathway of complement component C3 in the body, or ectopic expression or localization or accumulation. Regarding therapeutic use.

Double-stranded RNA (dsRNA) capable of complementarily binding expressed mRNA has been found to be capable of blocking gene expression by a mechanism called RNA interference (RNAi). (Fire et al., 1998, Nature. February 19, 1998; 391 (6669): 806-11 and Elbashir et al., 2001, Nature. May 24, 2001; 411 (6836): 494-8. page). Short dsRNAs have become a useful tool for inducing gene-specific post-transcriptional silencing and studying gene function in many organisms, including vertebrates. RNAi is mediated by RISC complex, a sequence-specific multi-component nuclease that degrades messenger RNA homologous to silence triggers loaded on RNA-induced silencing complex (RISC). Interfering RNAs such as siRNAs, antisense RNAs, and microRNAs (referred to herein as iRNAs) are gene silencing, i.e., by inhibiting gene translation of a protein by degrading mRNA molecules. It is an oligonucleotide that prevents formation. Gene silencing agents are becoming increasingly important for therapeutic applications in medicine.

According to Watts and Corey in the Journal of Pathology (2012, Vol 226, pp. 365-379), there are algorithms that can be used to design nucleic acid silencing triggers, all of which have severe limitations. Have. The algorithm does not take into account factors such as the tertiary structure of the target mRNA or the intervention of RNA-binding proteins, so various experimental methods can be used to identify potent iRNAs. Therefore, the discovery of potent nucleic acid silencing triggers with minimal off-target effects is a complex process. For drug development of these highly charged molecules, they need to be able to be economically synthesized, distributed in target tissues, enter cells and function within acceptable toxicity limits. Is.

The complement system or pathway is part of the innate immune system of host defense against invading pathogens. The complement system or pathway is mainly composed of a large number of proteins that circulate in the bloodstream in the form of precursors. Most of the proteins that form the complement system, including the complement component protein C3 (also simply referred to herein as C3), are synthesized by hepatocytes, primarily in the liver, and secreted into the bloodstream. To. System activation leads to an inflammatory response, leading to phagocytic attraction and opsonization, and thus elimination of pathogens, immune complexes and cell debris (Janeway's Immunobiology 9th Edition). The complement system consists of three pathways (classical pathway, leptin pathway and alternative pathway), all of which converge to the formation of the so-called complement component 3-convertase complex. These enzyme complexes cleave the complement component C3 protein into C3a and C3b. When cleaved, C3b now forms part of the complex that cleaves into C5a and C5b. After cleavage, C5b is one of the key components of the major complement pathway effector, which is a complement membrane attack complex. Therefore, C3 is an important component of the complement system activation pathway.

Several diseases are associated with abnormal acquired or genetic activation of the complement pathway, as well as overexpression or overexpression of C3. Among them, these are C3 glomerulonephritis (C3G), atypical hemolytic urinary toxicosis syndrome (aHUS), immune complex-mediated glomerulonephritis (IC-mediated GN), post-infection post-glomerulonephritis (PIGN), systemic. Sexual lupus erythematosus, lupus nephritis, ischemia-reperfusion injury and IgA nephropathy (as outlined in IgAN, Ricklin et al., Nephrology, 2016 and others). Most of these diseases are associated with the kidney. This is because this organ is incomparably sensitive to complement-induced injury. However, diseases of other organs also include age-related yellow spot degeneration (AMD), rheumatoid arthritis (RA), anti-neutrophil cytoplasmic autoantibody-related vasculitis (ANCA-AV), disbiosis periodontal disease, malaria anemia, It is known to be associated with complement dysfunction such as paroxysmal nocturnal hemoglobinuria (PNH) and septicemia.

In C3G, C3 accumulates in the glomeruli in the kidney and blocks them. Accumulation of C3 also leads to kidney damage. In atypical hemolytic urinary toxicosis syndrome (aHUS), the complement system targets erythrocytes, which cause erythrocyte lysis.

There are currently very few treatments for complement-mediated diseases, disorders and syndromes. The monoclonal humanized antibody eculizumab is one of them. Eculizumab is known to bind to the complement protein C5 and block the complement membrane attack complex at the ends of the complement cascade (Hillmen et al., 2006 NEJM). However, only some of the patients with the diseases listed above respond to eculizumab therapy. Therefore, there is an unmet, high need for medical treatment of complement-mediated or related diseases. C3 is a central factor in complement pathway activation. Therefore, inhibition of C3 presents promising therapeutic strategies for many complement-mediated diseases.

U.S. Pat. No. 5,885,968 European Patent No. 0 758 904 B1 International Publication No. 2017/174657

Fire et al., 1998, Nature. February 19, 1998; 391 (6669): 806-11 Elbashir et al., 2001, Nature. May 24, 2001; 411 (6836): 494-8 Watts and Corey in the Journal of Pathology (2012, Vol 226, pp. 365-379) Janeway's Immunobiology 9th Edition Ricklin et al., Nephrology, 2016 Hillmen et al., 2006 NEJM Dubber et al. (Bioconjug. Chem. 2003 Jan-Feb; 14 (1): 239-46) Weigel, P.H. et al., Biochim. Biophys. Acta. September 19, 2002; 1572 (2-3): pp. 341-63 Ishibashi, S. et al., J Biol. Chem. November 11, 1994; 269 (45): 27803-6 Biessen EA et al., J Med Chem. April 28, 1995; 38 (9): 1538-46 Akinc et al., Mol Ther. July 2010; 18 (7): 1357-64 Hoevelmann et al., Chem. Sci., 2016, 7, 128-135 Nair et al., J. Am. Chem. Soc., 2014, 136 (49), pp. 16958-1696. Prakash, Nucleic Acids Res., 2015, 43 (6), pp. 2993-3011 Haraszti, Nucleic Acids Res., 2017, 45 (13), pp. 7581-7592

One aspect of the present invention is a double-stranded nucleic acid for inhibiting the expression of complement component C3, wherein the nucleic acid comprises a first strand and a second strand, and the sequence of the first strand is , SEQ ID NOs: 361, 95, 111, 125, 131, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 , 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 , 89, 91, 93, 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 127, 129 or 133, and 3 or less nucleotides. A nucleic acid containing a sequence of at least 15 different nucleotides.

One aspect relates to a double-stranded nucleic acid capable of inhibiting the expression of complement component C3 for use as a pharmaceutical or in a related method.

One aspect is a composition comprising nucleic acids and delivery vehicles and / or physiologically acceptable excipients and / or carriers and / or diluents and / or buffers and / or preservatives disclosed herein. Regarding things.

One aspect relates to a composition comprising the nucleic acids disclosed herein and additional therapeutic agents selected from the group comprising oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies and peptides.

One aspect relates to the nucleic acids or compositions disclosed herein for use as pharmaceuticals or in related methods.

One aspect relates to the nucleic acids or compositions disclosed herein for use in the prevention of a disease, disorder or syndrome, reduction of the risk of suffering from a disease, disorder or syndrome, or in the treatment of a disease, disorder or syndrome. ..

One embodiment is the use of the nucleic acids or compositions disclosed herein in the prevention of a disease, disorder or syndrome, the reduction of the risk of suffering from a disease, disorder or syndrome, or the treatment of a disease, disorder or syndrome. , The disease, disorder or syndrome is preferably C3 glomerulopathy (C3G), with respect to use.

One embodiment comprises the step of administering a pharmaceutically effective dose of the nucleic acid or composition disclosed herein to an individual in need of treatment to prevent or prevent a disease, disorder or syndrome. , With respect to methods of reducing the risk of suffering from a disorder or syndrome, or treating a disease, disorder or syndrome, preferably where the nucleic acid or composition is administered to the subject, subcutaneously, intravenously or orally. , Administered by rectal, pulmonary or intraperitoneal administration.

The present invention relates to nucleic acids and compositions thereof that are double chains and are directed to RNA transcripts expressing complement component C3. These nucleic acids or conjugated nucleic acids or compositions can be used in the treatment and prevention of various diseases, disorders and syndromes for which reduced expression of the C3 gene product is desired.

A first aspect of the invention is a double-stranded nucleic acid, preferably for inhibiting the expression of C3 in cells, wherein the nucleic acid comprises a first strand and a second strand, said first. The sequences of the strands are SEQ ID NOs: 361, 95, 111, 125, 131, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, With any one of the sequences 83, 85, 87, 89, 91, 93, 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 127, 129 or 133. , Contains sequences of at least 15 nucleotides with 3 or less nucleotides different. These nucleic acids have the advantage of being active in various species associated with preclinical and clinical development, and / or having little associated off-target effect.

Preferably, the sequence of the first strand is SEQ ID NO: 361, 95, 111, 125, 131, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27. , 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77 , 79, 81, 83, 85, 87, 89, 91, 93, 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 127, 129 or 133 Any one of the above is different from 3 or less nucleotides, preferably 2 or less nucleotides, more preferably 1 or less nucleotides, and most preferably none of the nucleotides are different, at least 16, more preferably at least. It contains sequences of 17, more preferably at least 18, most preferably all 19 nucleotides.

Preferably, the sequence of the first strand of nucleic acid is SEQ ID NO: 361, 95, 111, 125, 131, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25. , 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 , 77, 79, 81, 83, 85, 87, 89, 91, 93, 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 127, 129 or 133 Consists of one of the arrays of. However, the sequence may be modified by a number of modifications that do not alter the uniqueness of the nucleotide. For example, modification of the nucleic acid skeleton does not change the uniqueness of the nucleotide, as the base itself remains the same as the reference sequence.

Nucleic acid comprising a sequence according to a reference sequence herein means that the nucleic acid comprises sequences of adjacent nucleotides in the order specified in the reference sequence.

References herein that include or consist of unmodified nucleotides are not limited to sequences that have unmodified nucleotides. The same reference also contains one, several, eg, 2, 3, 4, 5, 6, 7, 7 or more nucleotides, including all, 2'-OMe, 2'-F. , A ligand, a linker, the same nucleotide sequence modified by modifications such as 3'end or 5'end modification or any other modification. The same reference also refers to sequences in which two or more nucleotides are linked to each other by natural phosphodiester bonds or by any other bond, such as a phosphorothioate or phosphorodithioate bond.

Double-stranded nucleic acids are such that the first and second strands hybridize to each other over at least a portion of their length and therefore each strand under physiological conditions, eg, in PBS, at 37 ° C. It is a nucleic acid capable of forming a double-stranded region at a concentration of 1 μM. The first and second strands preferably hybridize to each other, thus forming a double-stranded region over a region of at least 15 nucleotides, preferably 16, 17, 18 or 19 nucleotides. It is possible to do. This double-stranded region comprises nucleotide base pairing between two strands, preferably based on Watson-Crick base pairing and / or wobble base pairing (eg, GU base pairing). All two strands of nucleotides within a double-stranded region do not pair with each other to form a double-stranded region. A certain number of mismatches, deletions or insertions between the nucleotide sequences of two strands are acceptable. An overhang on either end of either the first or second strand or an unpaired nucleotide at either end of a double-stranded nucleic acid is also possible. The double-stranded nucleic acid is preferably a stable double-stranded nucleic acid under physiological conditions, preferably at a concentration of 1 μM for each chain, preferably 45 ° C. or higher, preferably in PBS, for example. It has a melting temperature (Tm) of 50 ° C or higher, more preferably 55 ° C or higher.

Under physiological conditions, stable double-stranded nucleic acids are, for example, in PBS at a concentration of 1 μM for each chain, 45 ° C or higher, preferably 50 ° C or higher, more preferably 55 ° C. Or a double-stranded nucleic acid having a higher Tm.

The first and second strands are preferably i) over a portion of their length, preferably over at least 15 nucleotides in both of those lengths, ii) the entire length of the first strand. Over, iii) over the entire length of the second strand, or iv) over the entire length of both the first and second strands, it is possible to form double-stranded regions (ie, each other). Complementary). Chains that are complementary to each other over a particular length mean that the chains can base pair to each other over either Watson-click or wobble base pairing. do. Each length of nucleotide can base pair with its counterpart in another strand over a given length, as long as stable double-stranded nucleotides can be formed under physiological conditions. It does not have to be. However, each nucleotide of length is preferred when base pairing with its counterpart in another strand over a given length.

A certain number of mismatches, deletions or insertions between the first strand and the target sequence, or between the first strand and the second strand, are acceptable in the context of siRNA. In addition, in certain cases, it may increase RNA interference (eg, inhibition) activity.

Nucleic acid inhibitory activity according to the invention depends on the formation of a double-stranded region between all or part of the first strand and part of the target nucleic acid. Starting with the first base pair formed between the first strand and the target sequence and ending with the last base pair formed between the first strand and the target sequence (including both ends). The portion of the defined target nucleic acid that forms the first strand and the double strand region is the target nucleic acid sequence, or simply the target sequence. The double-stranded region formed between the first strand and the second strand need not be the same as the double-stranded region formed between the first strand and the target sequence. That is, the second strand may have a sequence different from the target sequence, but the first strand has a double-stranded structure having both the second strand and the target sequence, at least under physiological conditions. It must be possible to form.

Complementarity between the first strand and the target sequence may be complete (ie, without nucleotide mismatch or insertion or deletion in the first strand when compared to the target sequence. 100% identity).

The complementarity between the first strand and the target sequence does not have to be perfect. Complementarity can be from about 70% to about 100%. More specifically, the complementarity may be at least 70%, 80%, 85%, 90% or 95% and an intermediate value thereof.

The identity between the first strand and the complementary sequence of the target sequence can range from about 75% to about 100%. More specifically, complementarity is at least 75%, 80%, 85%, 90% or 95% and their median if the nucleic acid is capable of reducing or inhibiting the expression of complement component C3. May be.

Nucleic acid with less than 100% complementarity between the first strand and the target sequence complements to the same level as nucleic acid with complete complementarity between the first strand and the target sequence. It may be possible to reduce the expression of component C3. Alternatively, a nucleic acid having less than 100% complementarity between the first strand and the target sequence will reduce the expression of complement component C3 by 15% of the level of reduction achieved by the nucleic acid with full complementarity. It may be possible to reduce to a level of ~ 100%.

In one aspect, the nucleic acids of the present disclosure are
(a) The sequence of the first strand contains any one of the sequences of the first strand in Table 1 and a sequence in which no more than three nucleotides are different, and optionally the sequence of the second strand. Whether the sequence contains a sequence in the second strand of the same row in the table and a sequence in which no more than 3 nucleotides are different.
(b) The sequence of the first strand contains any one of the sequences of the first strand in Table 1 and a sequence in which no more than two nucleotides are different, and optionally the sequence of the second strand. Whether the sequence contains a sequence in the second strand of the same row in the table and a sequence in which no more than two nucleotides are different.
(c) The sequence of the first strand contains any one of the sequences of the first strand in Table 1 and a sequence in which one or less nucleotides are different, and optionally, of the second strand. Does the sequence contain a sequence that differs from the sequence in the second strand of the same row in the table by one or less nucleotides?
(d) The sequence of the first strand contains any one of the sequences of the first strand in Table 1 and, optionally, the sequence of the second strand is in the same row of the table. Contains or contains a sequence of sequences of the second strand
(e) The sequence of the first strand consists of any one of the sequences of the first strand in Table 1 and, optionally, the sequence of the second strand is the first in the same row of the table. A nucleic acid consisting of a sequence of two strands, Table 1 shows:

In one aspect, the nucleic acid is
(a) The sequence of the first strand contains the sequence of SEQ ID NO: 361 and, optionally, the sequence of the second strand contains the sequence of SEQ ID NO: 112, or
(b) The sequence of the first strand contains the sequence of SEQ ID NO: 95 and, optionally, the sequence of the second strand contains the sequence of SEQ ID NO: 96 or
(c) The sequence of the first strand contains the sequence of SEQ ID NO: 111 and, optionally, the sequence of the second strand contains the sequence of SEQ ID NO: 112 or
(d) The sequence of the first strand contains the sequence of SEQ ID NO: 125 and, optionally, the sequence of the second strand contains the sequence of SEQ ID NO: 126 or
(e) The sequence of the first strand contains the sequence of SEQ ID NO: 131 and, optionally, the sequence of the second strand contains the sequence of SEQ ID NO: 132 or
(f) The sequence of the first strand consists of SEQ ID NO: 361 and, optionally, the sequence of the second strand consists of SEQ ID NO: 112, or
(g) The sequence of the first strand consists of SEQ ID NO: 95 and, optionally, the sequence of the second strand consists of SEQ ID NO: 96, or
(h) The sequence of the first strand consists of SEQ ID NO: 111 and, optionally, the sequence of the second strand consists of SEQ ID NO: 112, or
(i) The sequence of the first strand consists of SEQ ID NO: 125 and, optionally, the sequence of the second strand consists of SEQ ID NO: 126, or
(j) The sequence of the first strand is a nucleic acid consisting of SEQ ID NO: 131 and, optionally, the sequence of the second strand is a nucleic acid consisting of SEQ ID NO: 132.

In one aspect, if the 5'endmost nucleotide of the first strand is a nucleotide other than A or U, this nucleotide will be replaced with A or U in the sequence. Preferably, if the 5'endmost nucleotide of the first strand is a nucleotide other than U, this nucleotide is replaced with U in the sequence, more preferably with U using 5'-vinylphosphonate.

The nucleic acids of the invention do not contain the entire sequence of reference first and / or second strand sequences, eg, as conferred in Table 1, or one or both strands are equivalent. If the reference sequence to be used differs from one, two or three nucleotides, the nucleic acid is preferably the corresponding nucleic acid, including the entire reference sequence of the first and second strands in comparable experiments. At least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 80%, still more preferably at least 90%, even more preferably at least 95% of the inhibitory activity of C3. Hold%, most preferably 100%.

In one embodiment, the nucleic acid comprises the sequence of SEQ ID NO: 361, or preferably the sequence of SEQ ID NO: 361, of which the sequence of the first strand comprises, and optionally, the sequence of SEQ ID NO: 112. At least 15, preferably at least 16, more preferably at least 17, more preferably at least 18, most preferably containing all nucleotides, or preferably at least 15 of the sequence of SEQ ID NO: 112. It preferably consists of at least 16, more preferably at least 17, even more preferably at least 18, most preferably all nucleotides, or the sequence of the first strand comprises or preferably contains the sequence of SEQ ID NO: 95. Consists of the sequence of SEQ ID NO: 95, and optionally, the sequence of the second strand is at least 15, preferably at least 16, more preferably at least 17, and even more preferably at least 18 of the sequence of SEQ ID NO: 96. , Most preferably all nucleotides, or preferably at least 15 of the sequence of SEQ ID NO: 96, preferably at least 16, more preferably at least 17, still more preferably at least 18, and most preferably all nucleotides. Consists of, or the sequence of the first strand comprises the sequence of SEQ ID NO: 111, or preferably consists of the sequence of SEQ ID NO: 111, and optionally, the sequence of the second strand consists of the sequence of SEQ ID NO: 112. At least 15, preferably at least 16, more preferably at least 17, even more preferably at least 18, most preferably containing all the nucleotides, or preferably at least 15 of the sequence of SEQ ID NO: 112, preferably. Consists of at least 16, more preferably at least 17, even more preferably at least 18, most preferably all nucleotides, or the sequence of the first strand comprises or preferably sequences of SEQ ID NO: 125. Consisting of the sequence of number 125, optionally, the sequence of the second strand is at least 15, preferably at least 16, more preferably at least 17, and even more preferably at least 18, most of the sequence of SEQ ID NO: 126. It contains all the nucleotides, or preferably consists of at least 15 of the sequence of SEQ ID NO: 126, preferably at least 16, more preferably at least 17, still more preferably at least 18, and most preferably all nucleotides. mosquito, Alternatively, the sequence of the first strand comprises or preferably consists of the sequence of SEQ ID NO: 131, and optionally, the sequence of the second strand contains at least 15 of the sequences of SEQ ID NO: 132. , Preferably at least 16, more preferably at least 17, still more preferably at least 18, and most preferably at least 15 of the sequence of SEQ ID NO: 132, preferably at least 16. A nucleic acid consisting of at least 17, more preferably at least 18, and most preferably all nucleotides.

In one aspect, the nucleic acid is a double-stranded nucleic acid, preferably for inhibiting expression of C3 in cells, wherein the nucleic acid comprises a first nucleic acid strand and a second nucleic acid strand, the first. The strands are SEQ ID NOs: 112, 96, 126, 132, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38. , 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88 , 90, 92, 94, 98, 100, 102, 104, 106, 108, 110, 114, 116, 118, 120, 122, 124, 128, 130 or 134 sequence nucleic acid hybridized under physiological conditions. It is possible to soy and the second strand can hybridize to the first strand under physiological conditions to form a double-stranded region.

Nucleic acids capable of hybridizing under physiological conditions are base pairs, preferably Watson-click or wobble bases, between at least some of the opposite nucleotides in the strand so as to form at least a double-stranded region. Nucleic acids that can form pairs. Such double-stranded nucleic acids are preferably stable double-stranded nucleic acids under physiological conditions (eg, in PBS at a concentration of 1 μM each chain at 37 ° C.), under such conditions two strands. Means that they remain hybridized to each other. The Tm of the double chain nucleotide is preferably 45 ° C. or higher, preferably 50 ° C. or higher, and more preferably 55 ° C. or higher.

One embodiment relates to a nucleic acid for inhibiting the expression of complement component C3, wherein the nucleic acid is any of the sequences in Table 5 and 3 or less nucleotides, preferably 2 or less nucleotides. More preferably one or less nucleotides differ, and most preferably none of the nucleotides differ, at least 15, preferably at least 16, more preferably at least 17, still more preferably at least 18, and most preferably all. Containing the first sequence of nucleotides, the first sequence is capable of hybridizing to a target gene transcript (eg, mRNA) under physiological conditions. Preferably, the nucleic acid differs from any of the sequences in Table 5 with 3 or less nucleotides, preferably 2 or less nucleotides, more preferably 1 or less nucleotides, and most preferably any nucleotide. No difference, at least 15, preferably at least 16, more preferably at least 17, even more preferably at least 18, further comprising a second sequence of all nucleotides, the second sequence being physiological. Under conditions, it is possible to hybridize to the first sequence, preferably the nucleic acid is a siRNA capable of inhibiting C3 expression via the RNAi pathway.

One aspect relates to any double-stranded nucleic acid as disclosed in Table 3 for inhibiting the expression of complement component C3. These nucleic acids are all siRNAs with various nucleotide modifications. Some of them are conjugates containing GalNAc moieties that can specifically target cells with GalNAc receptors, such as hepatocytes.

One aspect relates to a double-stranded nucleic acid capable of inhibiting the expression of complement component C3, preferably for use as a pharmaceutical.

The nucleic acids described herein may be capable of inhibiting the expression of complement component C3. The inhibition may be complete, i.e., the residual expression can be 0% as compared to the expression level of C3 in the absence of the nucleic acids of the invention. Inhibition of C3 expression may be partial, i.e., inhibition of C3 expression is 15%, 20%, 30%, 40%, 50% of C3 expression in the absence of the nucleic acids of the invention. It can be 60%, 70%, 75%, 80%, 85%, 90%, 95% or more, or an intermediate value. The level of inhibition can be measured by comparing the treated sample with an untreated sample or, for example, a sample treated with a control such as siRNA that does not target C3. Inhibition can be measured by measuring C3 mRNA and / or protein levels, or levels of biomarkers or indicators that correlate with the presence or activity of C3. Inhibition can be measured in cells that may have been treated in vitro with the nucleic acids described herein. Alternatively, or further, the inhibition is in cells such as hepatocytes, or in tissues such as liver tissue, or in organs such as liver, or in body fluids such as blood, serum, lymph or nucleic acids disclosed herein. It can be measured in any other body part taken from the subject treated up to. Preferably, inhibition of C3 expression is 24 hours or 48 hours under ideal conditions (see Examples for appropriate concentrations and conditions) with the double-stranded RNA disclosed herein. C3 mRNA levels measured in C3-expressing cells after in vitro treatment with C3 mRNA levels measured in the same cells that were untreated, sham-treated, or treated with a control duplex. Determined by comparison.

One aspect of the invention is a first strand such that the first strand and the second strand can hybridize to each other, thereby forming a double-stranded nucleic acid having a double-stranded region. And the second strand relates to a nucleic acid that resides on a single strand of the nucleic acid that loops around it.

Preferably, the first and second strands of the nucleic acid are separate strands. The two separate strands are preferably 17 nucleotides to 25 nucleotides in length, more preferably 18 nucleotides to 25 nucleotides in length, respectively. The two chains can be the same length or different lengths. The first strand may be 17 to 25 nucleotides in length, preferably the first strand may be 18 to 24 nucleotides in length, and the first strand may be 18, 19, It may be 20, 21, 22, 23 or 24 nucleotides in length. Most preferably, the first strand is 19 nucleotides in length. The second strand may independently be 17 to 25 nucleotides in length, preferably the second strand may be 18 to 24 nucleotides in length, and the second strand may be 18 to 24 nucleotides in length. It may be 18, 19, 20, 21, 22, 23 or 24 nucleotides in length. More preferably, the second strand is 18 or 19 nucleotides in length, and most preferably the second strand is 19 nucleotides in length.

Preferably, the first and second strands of nucleic acid form double-stranded regions 17 and 25 nucleotides in length. More preferably, the double-stranded region is 18 to 24 nucleotides in length. The double-stranded region may be 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In the most preferred embodiment, the double-stranded region is 18 nucleotides in length. The double-stranded region is here the first strand that is base-paired to the nucleotide of the second strand from the 5'endmost nucleotide of the first strand that is base-paired to the nucleotide of the second strand. The region between the 3'end of the nucleotide and the 5'end of the first strand which is base paired with the nucleotide of the second strand and the nucleotide of the second strand is paired with the nucleotide of the second strand. Includes the 3'endmost nucleotide of a chain). The double-stranded region may contain nucleotides in one or both strands that are not base paired with respect to the nucleotides in the other strand. The double-stranded region may contain one, two, three or four such nucleotides on the first and / or second strand. However, preferably, the double-stranded region consists of 17 to 25 consecutive nucleotide base pairs. That is, the double-stranded region preferably comprises 17-25 contiguous nucleotides on both strands that are all base paired to the nucleotides in the other strand. More preferably, the double-stranded region consists of 18 or 19, most preferably 18 consecutive nucleotide base pairs.

In each of the embodiments disclosed herein, the nucleic acid may have a blunt end at both ends, an overhang at one end, and a blunt end at the other end. , Or may have overhangs at both ends.

Nucleic acids may have an overhang at one end and a blunt end at the other end. Nucleic acid may have overhangs at both ends. The nucleic acid may be blunt at both ends. Nucleic acids are blunt-ended at the 5'end of the first strand and the 3'end of the second strand, or at the 3'end of the first strand and the 5'end of the second strand. May be good.

Nucleic acids may contain overhangs at the 3'end or 5'end. The nucleic acid may have a 3'overhang on the first strand. The nucleic acid may have a 3'overhang on the second strand. The nucleic acid may have a 5'overhang on the first strand. The nucleic acid may have a 5'overhang on the second strand. Nucleic acids may have overhangs at both the 5'and 3'ends of the first strand. The nucleic acid may have overhangs at both the 5'and 3'ends of the second strand. The nucleic acid may have a 5'overhang on the first strand and a 3'overhang on the second strand. The nucleic acid may have a 3'overhang on the first strand and a 5'overhang on the second strand. The nucleic acid may have a 3'overhang on the first strand and a 3'overhang on the second strand. The nucleic acid may have a 5'overhang on the first strand and a 5'overhang on the second strand.

Overhangs at the 3'or 5'ends of the second or first strand can consist of 1, 2, 3, 4 and 5 nucleotides in length. Optionally, the overhang can consist of one or two nucleotides that can or cannot be modified.

In one embodiment, the 5'end of the first strand is a single strand overhang of one, two or three nucleotides, preferably one nucleotide.

Preferably, the nucleic acid is siRNA. siRNA is a small interfering or small silencing RNA that can inhibit the expression of a target gene by the RNA interference (RNAi) pathway. Inhibition occurs after transcription and after targeted degradation of the mRNA transcript of the target gene. siRNAs form part of the RISC complex. The RISC complex specifically targets the target RNA by sequence complementarity of the first (antisense) strand having the target sequence.

Preferably, the nucleic acid mediates RNA interference (RNAi). Preferably, the nucleic acid is at least 50%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95% inhibitory, most preferably 100% inhibitory. Sex mediates RNA interference. Therefore, the nucleic acid, or at least the first strand of nucleic acid, can preferably be incorporated into the RISC complex. Thus, as a result, the nucleic acid, or at least the first strand of the nucleic acid, can lead the RISC complex to the nucleic acid, or a specific target RNA in which at least the first strand of the nucleic acid is at least partially complementary. It is possible. Subsequently, the RISC complex specifically cleaves this target RNA, resulting in inhibition of expression of the gene from which the RNA is derived.

Nucleic Acid Modifications Nucleic acid modifications of the invention generally provide powerful tools for overcoming potential limitations including, but not limited to, in vitro and in vivo stability as well as bioavailability inherent in natural RNA molecules. offer. The nucleic acid according to the invention can be modified by chemical modification. In addition, the modified nucleic acid can minimize the possibility of inducing interferon activity in humans. Modifications can further enhance the functional delivery of nucleic acids to target cells. The modified nucleic acid of the invention may comprise one or more chemically modified ribonucleotides of one or both of the first and second strands. Ribonucleotides can include chemical modifications of bases, sugars or phosphate moieties. Ribonucleic acid can be modified by substitution with a nucleic acid or base analog or insertion of a nucleic acid or base analog.

Throughout the description of the invention, the "same modification or common modification" is A, G, C or U modified with a group such as a methyl group (2'-OMe) or a fluoro group (2'-F). Means the same modification to any nucleotide, such as. For example, 2'-F-dU, 2'-F-dA, 2'-F-dC, 2'-F-dG are all 2'-OMe-rU, 2'-OMe-rA, 2'-OMe. Considered to be the same or common modification, such as -rC, 2'-OMe-rG. In contrast, the 2'-F modification is a different modification compared to the 2'-OMe modification.

Preferably, at least one nucleotide in the first and / or second strand of the nucleic acid is a modified nucleotide, preferably a non-naturally occurring nucleotide, eg, preferably a 2'-F modified nucleotide.

The modified nucleotide can be a nucleotide having a modification of a glycosyl group. The 2'hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents.

"Oxy" -2-As an example of hydroxyl modification, alkoxy or aryloxy (OR, eg, R = H, alkyl (eg, methyl), cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycol (PEG) , O (CH2CH2O) nCH2CH2OR; "Locked" nucleic acid (LNA) where the 2'hydroxyl is attached to the 4'carbon of the same ribose sugar, for example by methylene cross-linking; O-AMINE (AMINE = NH2, alkylamino, dialkylamino, Heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O (CH2) nAMINE (eg AMINE = NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, Diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino).

As "deoxy" modifications, hydrogen, halogen, amino (eg NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acids); NH (CH2CH2NH) nCH2CH2-AMINE (AMINE = NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino), -NHC (O) R (R = alkyl, cycloalkyl, aryl, aralkyl, heteroaryl Or sugars), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may optionally be substituted with, for example, amino-functionality. .. Other substituents of certain embodiments include 2'-methoxymethyl, 2'-OCH3, 2'-O-allyl, 2'-C-allyl, and 2'-fluoro.

Glycosyls can also contain one or more carbons that have the opposite stereochemical configuration of the corresponding carbon in ribose. Therefore, the modified nucleotide may contain sugars such as arabinose.

Modified nucleotides can also contain "debase" sugars, which lack the nucleobase found in C-1'. These debased sugars may further contain modifications at one or more of the constituent sugar atoms.

The 2'modification can be used in combination with one or more phosphate linker modifications (eg, phosphorothioates).

One or more nucleotides of the nucleic acid of the invention can be modified. The nucleic acid may contain at least one modified nucleotide. The modified nucleotide can be in the first strand. The modified nucleotide can be in the second strand. The modified nucleotide can be in the double-stranded region. The modified nucleotide can be located outside the double-stranded region, i.e., in the single-stranded region. The modified nucleotide may be on the first strand or outside the double-stranded region. The modified nucleotide may be on the second strand or outside the double strand region. The 3'end nucleotide of the first strand may be a modified nucleotide. The 3'end nucleotide of the second strand may be a modified nucleotide. The 5'end nucleotide of the first strand may be a modified nucleotide. The 5'end nucleotide of the second strand may be a modified nucleotide.

The nucleic acid of the present invention may have one modified nucleotide, or the nucleic acid of the present invention may have about 2 to 4 modified nucleotides, or 4 to 6 nucleic acids. Modified nucleotides, about 6 to 8 modified nucleotides, about 8 to 10 modified nucleotides, about 10 to 12 modified nucleotides, about 12 to 14 modified nucleotides, about 14 to 16 Modified nucleotides, about 16 to 18 modified nucleotides, about 18 to 20 modified nucleotides, about 20 to 22 modified nucleotides, about 22 to 24 modified nucleotides, about 24 to 26 modified nucleotides. May have modified nucleotides of, or about 26-28 modified nucleotides. In each case, the nucleic acid containing the modified nucleotide is the same, but retains at least 50% of its activity as compared to the nucleic acid without the modified nucleotide and vice versa. Nucleic acids are the same, but 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of their activity when compared to nucleic acids without the above modified nucleotides. Alternatively, it may retain 100% and an intermediate value, or it may have more activity than 100% of the nucleic acid without the modified nucleotides.

The modified nucleotide can be a purine or a pyrimidine. At least half of the pudding may be modified. At least half of the pyrimidines may be modified. All puddings may be modified. All pyrimidines may be modified. Modified nucleotides include 3'terminal deoxytimine (dT) nucleotides, 2'-O-methyl (2'-OMe) modified nucleotides, 2'modified nucleotides, 2'deoxy modified nucleotides, locked nucleotides, debase nucleotides, 2'. Includes amino-modified nucleotides, 2'alkyl-modified nucleotides, 2'-deoxy-2'-fluoro (2'-F) -modified nucleotides, morpholinonucleotides, phosphoramidates, nucleotides containing unnatural bases, 5'-phosphorothioate groups It can be selected from the group consisting of nucleotides, nucleotides containing 5'phosphates or 5'phosphate mimetics and terminal nucleotides linked to cosleteryl derivatives or dodecanoic acid bisdecylamide groups.

The nucleic acid may include a nucleotide containing a modified base, wherein the base is 2-aminoadenosine, 2,6-diaminopurine, inosin, pyridine-4-one, pyridine-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (eg 5-methylcytidine), 5-alkyluridine (eg ribothymidine), 5-halolysine (eg) For example, 5-bromouridine), 6-azapyrimidine, 6-alkylpyrimidine (eg, 6-methyluridine), propine, quesosine, 2-thiouridine, 4-thiouridine, wibutosine, wipetoxocin, 4-acetylcytidine, 5- (Carboxyhydroxymethyl) uridine, 5'-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosyleocin, 1-methyladenosine, 1-methylinosin, 2,2 -Dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonyl It is selected from methyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosyleosin, uridine-5-oxyacetic acid and 2-thiocytidine.

Nucleic acids discussed herein include unmodified RNA as well as RNA modified to improve efficacy or stability, for example. Unmodified RNA is a molecule in which the components of the nucleic acid, ie, sugars, bases and phosphate moieties, are the same as or essentially the same as naturally occurring, eg, as naturally occurring in the human body. Point to. As used herein, the term "modified nucleotide" refers to a nucleotide component, i.e., one or more of a sugar, a base, and a phosphate moiety, which is different from the naturally occurring nucleotide. The term "modified nucleotide" is also rigorous because, in certain cases, the modified nucleotide lacks or has an alternative to the essential component of the nucleotide, such as a sugar, base or phosphate moiety. In a sense, it refers to a molecule that is not a nucleotide. It is understood that a nucleic acid containing such a modified nucleotide will still be a nucleic acid, even if one or more of the nucleotides of the nucleic acid are missing or replaced by a modified nucleotide having an essential component of the nucleotide. To.

Many of the modifications made herein within the nucleic acid, such as modifications of the base, or the phosphate moiety, or the unlinked O of the phosphate moiety, are repeated within the polynucleotide molecule. In some cases, the modification is done at all possible positions / nucleotides in the polynucleotide, but in many cases no modification is done. Modifications may be made only at the 3'or 5'end positions, at the end regions such as positions on the end nucleotides, or at the last two, three, four, five, or ten chains. It may be done only in nucleotides. Modifications may be made in the double chain region, the single chain region, or both. The modification may be carried out only in the double chain region of the nucleic acid of the present invention, or may be carried out only in the single chain region of the nucleic acid of the present invention. Phosphorothioate modification at the unconnected O position may be performed at only one or both ends and at the terminal region, eg, at a position on the terminal nucleotide, or at the last two, three, four, or five of the strands. It may be done only on a single nucleotide, or in a double-stranded region and / or in a single-stranded region, especially at the terminal. The 5'end and / or the 3'end can be phosphorylated.

The stability of the nucleic acids of the invention is increased by the inclusion of a specific base in the overhang, or by the inclusion of modified nucleotides in a single chain overhang, eg, in a 5'and 3'overhang, or both. Can be done. Purine nucleotides can be included in the overhang. All or some of the bases in the 3'or 5'overhang may be modified. Modifications may include the use of modifications at the 2'-OH group of ribose sugars, the use of deoxyribonucleotides in place of ribonucleotides, and modifications at phosphate groups such as phosphorothioate modifications. The overhang does not have to be homologous to the target sequence.

The nuclease can hydrolyze nucleic acid phosphodiester bonds. However, chemical modifications to nucleic acids can confer improved properties and make oligoribonucleotides more stable to nucleases.

Modified nucleic acids, as used herein, may include one or more of the following:
(i) One or more modifications of one or both of the unlinked phosphate oxygen and / or one or more modifications of the linked phosphate oxygen (referred to as linked, even if present at the 5'and 3'ends of the nucleic acids of the invention), eg. , Replace;
(ii) Modification of 2'hydroxyl on ribose sugar components, eg ribose sugar, eg replacement;
(iii) Replacement of the phosphate moiety with a "dephospho"linker;
(iv) Modification or replacement of naturally occurring bases;
(v) Ribose-replacement or modification of the phosphate skeleton, as well as
(vi) Modification of the 3'end or 5'end of the first and / or second strand, eg, removal, modification or replacement or moiety of the terminal phosphate group, eg, one of the fluorescently labeled moieties or Conjugation to either the 3'or 5'end of both strands.

The terms replacement, modification, and modification refer to differences from naturally occurring molecules.

The specific modifications are discussed in detail below.

The nucleic acid may contain one or more nucleotides on the modified second and / or first strand.

Alternate means, as described herein, appearing one after another in a regular manner. In other words, alternating means that they appear repeatedly in sequence. For example, when one nucleotide is modified, the next adjacent nucleotide is not modified, the following adjacent nucleotide is modified, and so on. If one nucleotide can be modified with the first modification, the next adjacent nucleotide may be modified with the second modification, and the subsequent adjacent nucleotides may be modified with the first modification. , Etc., where the first and second modifications are different.

Some representative modified nucleic acid sequences of the invention are shown in Examples. These examples are intended to be representative and non-limiting.

In one aspect of the nucleic acid, at least nucleotides 2 and 14 of the first strand are preferably modified by the first common modification, starting with nucleotide number 1 at the 5'end of the first strand. Numbered consecutively. The first modification is preferably 2'-F.

In one embodiment, at least one, some or preferably all of the even numbered nucleotides of the first strand are preferably modified by the first common modification and the nucleotides are 5 of the first strand. 'The numbers start with nucleotide number 1 at the end and are numbered consecutively. The first modification is preferably 2'-F.

In one embodiment, at least one, some or preferably all of the odd numbered nucleotides of the first strand are modified, starting with nucleotide number 1 at the 5'end of the first strand. Numbered consecutively. Preferably, the nucleotide is modified by a second modification. If the nucleic acid also contains, for example, the first modification of nucleotides 2 and 14 of the first strand, or of all the nucleotides numbered even, this second modification is with the first modification. It is preferable that they are different. The first modification is preferably any 2'ribose modification, or locked nucleic acid (LNA), or non-2'-OH group having the same volume as the 2'-OH group or having a smaller volume than the 2'-OH group. It is a locked nucleic acid (UNA) or 2'-fluoroarabino nucleic acid (FANA) modification. A 2'ribose modification with the same volume as the 2'-OH group or a smaller volume than the 2'-OH group can be, for example, 2'-F, 2'-H, 2'-halo, or 2'-OH. '-Can be NH ₂ . The second modification is preferably any 2'ribose modification with a larger volume than the 2'-OH group. A 2'ribose modification with a larger volume than the 2'-OH group is, for example, 2'-OMe, 2'-O-MOE (2'-O-methoxyethyl), 2'-O-allyl or 2'-O. -Although it can be alkyl, the nucleic acid is capable of reducing the expression of the target gene under comparable conditions to at least to the same extent as the same but unmodified nucleic acid. The first modification is preferably 2'-F and / or the second modification is preferably 2'-OMe.

In one embodiment, at least one, some, or preferably all of the nucleotides in the second strand at positions corresponding to the even numbered nucleotides in the first strand are preferably modified by the third modification. To. Preferably, in the same nucleic acid, nucleotide 2 of the first strand and all nucleotides numbered 14 or even are modified with the first modification. Further, or / or, the odd numbered nucleotides of the first strand are modified with the second modification. Preferably, the third modification is different from the first modification and / or the third modification is the same as the second modification. The first modification is preferably any 2'ribose modification, or locked nucleic acid (LNA), or unlocked, which is preferably of the same volume as the 2'-OH group or has a smaller volume than the 2'-OH group. Nucleic acid (UNA) or 2'-fluoroarabino nucleic acid (FANA) modifications. A 2'ribose modification with the same volume as the 2'-OH group or a smaller volume than the 2'-OH group can be, for example, 2'-F, 2'-H, 2'-halo, or 2'-OH. '-Can be NH ₂ . The second and / or third modification is preferably any 2'ribose modification with a larger volume than the 2'-OH group. A 2'ribose modification with a larger volume than the 2'-OH group is, for example, 2'-OMe, 2'-O-MOE (2'-O-methoxyethyl), 2'-O-allyl or 2'-O. -Although it can be alkyl, the nucleic acid is capable of reducing the expression of the target gene under comparable conditions to at least to the same extent as the same but unmodified nucleic acid. The first modification is preferably 2'-F, and / or the second and / or third modification is preferably 2'-OMe. Nucleotides on the first strand are numbered sequentially starting with nucleotide number 1 at the 5'end of the first strand.

For example, a second strand nucleotide located at a position corresponding to an even numbered nucleotide in the first strand is paired with an even numbered nucleotide in the first strand. It is a nucleotide of the chain.

In one aspect, at least one, some or preferably all of the nucleotides in the second strand at the positions corresponding to the odd numbered nucleotides in the first strand are preferably modified by the fourth modification. .. Preferably, in the same nucleic acid, nucleotide 2 of the first strand and all nucleotides numbered 14 or even are modified with the first modification. Further, or / or, the odd numbered nucleotides of the first strand are modified with the second modification. Further, or / or, all the nucleotides of the second strand at the positions corresponding to the even numbered nucleotides of the first strand are modified by the third modification. The fourth modification is preferably different from the second modification, preferably different from the third modification, and the fourth modification is preferably the same as the first modification. The first and / or fourth modification is preferably any 2'ribose modification, or locked nucleic acid (LNA), having the same volume as the 2'-OH group or smaller in volume than the 2'-OH group. ), Or non-locked nucleic acid (UNA), or 2'-fluoroarabinonucleic acid (FANA) modification. A 2'ribose modification with the same volume as the 2'-OH group or a smaller volume than the 2'-OH group can be, for example, 2'-F, 2'-H, 2'-halo, or 2'-OH. '-Can be NH ₂ . The second and / or third modification is preferably any 2'ribose modification with a larger volume than the 2'-OH group. A 2'ribose modification with a larger volume than the 2'-OH group is, for example, 2'-OMe, 2'-O-MOE (2'-O-methoxyethyl), 2'-O-allyl or 2'-O. -Although it can be alkyl, the nucleic acid is capable of reducing the expression of the target gene under comparable conditions to at least to the same extent as the same but unmodified nucleic acid. The first and / or fourth modification is preferably a 2'-OMe modification, and / or the second and / or third modification is preferably a 2'-F modification. Nucleotides on the first strand are numbered sequentially starting with nucleotide number 1 at the 5'end of the first strand.

In one embodiment of the nucleic acid, the second strand nucleotide (s) at positions corresponding to nucleotides 11 or 13 or nucleotides 11 and 13 or nucleotides 11-13 of the first strand are modified by the fourth modification. Will be done. Preferably, all nucleotides in the second strand other than nucleotides 11 or 13 in the first strand or nucleotides 11 and 13 or nucleotides 11-13 at positions corresponding to nucleotides 11-13 are all by the third modification. Be qualified. Preferably, in the same nucleic acid, nucleotide 2 of the first strand and all nucleotides numbered 14 or even are modified with the first modification. Further, or / or, the odd numbered nucleotides of the first strand are modified with the second modification. The fourth modification is preferably different from the second modification, preferably different from the third modification, and the fourth modification is preferably the same as the first modification. The first and / or fourth modification is preferably any 2'ribose modification, or locked nucleic acid (LNA), having the same volume as the 2'-OH group or smaller in volume than the 2'-OH group. ), Or non-locked nucleic acid (UNA), or 2'-fluoroarabinonucleic acid (FANA) modification. A 2'ribose modification with the same volume as the 2'-OH group or a smaller volume than the 2'-OH group can be, for example, 2'-F, 2'-H, 2'-halo, or 2'-OH. '-Can be NH ₂ . The second and / or third modification is preferably any 2'ribose modification with a larger volume than the 2'-OH group. A 2'ribose modification with a larger volume than the 2'-OH group is, for example, 2'-OMe, 2'-O-MOE (2'-O-methoxyethyl), 2'-O-allyl or 2'-O. -Although it can be alkyl, the nucleic acid is capable of reducing the expression of the target gene under comparable conditions to at least to the same extent as the same but unmodified nucleic acid. The first and / or fourth modification is preferably a 2'-OMe modification, and / or the second and / or third modification is preferably a 2'-F modification. Nucleotides on the first strand are numbered sequentially starting with nucleotide number 1 at the 5'end of the first strand.

In one aspect of nucleic acid, all even numbered nucleotides in the first strand are modified by the first modification and all odd numbered nucleotides in the first strand are modified by the second modification. All of the nucleotides in the second strand at the positions corresponding to the even numbered nucleotides in the first strand are modified by the third modification and correspond to the odd numbered nucleotides in the first strand. All nucleotides in the second strand at the position where it is modified are modified by the fourth modification, where the first and / or fourth modification is 2'-F and / or the second and / or the second. The modification of 3 is 2'-OMe.

In one embodiment of the nucleic acid, all the even numbered nucleotides of the first strand are modified by the first modification and all the odd numbered nucleotides of the first strand are modified by the second modification. Then, all the nucleotides of the second strand at the positions corresponding to the nucleotides 11 to 13 of the first strand are modified by the fourth modification, and the second except the nucleotides corresponding to the nucleotides 11 to 13 of the first strand. All nucleotides in the strand of are modified by the third modification, where the first and fourth modifications are 2'-F and the second and third modifications are 2'-OMe. Preferably, in this embodiment, the 3'end nucleotide of the second strand is a reverse RNA nucleotide (ie, the nucleotide is not due to its 5'carbon, which would normally be, but by its 3'carbon. Connected to the 3'end of the chain). If the 3'end nucleotide of the second strand is a reverse RNA nucleotide, the reverse RNA nucleotide is an unmodified nucleotide in the sense that it does not contain any modifications as compared to the native nucleotide counterpart. Is preferable. Specifically, the reverse RNA nucleotide is preferably a 2'-OH nucleotide. Preferably, in this embodiment, if the 3'end nucleotide of the second strand is a reverse RNA nucleotide, the nucleic acid is blunt-ended at the end comprising at least the 5'end of the first strand.

One aspect of the invention is a nucleic acid as disclosed herein, preferably for inhibiting expression of the C3 gene in cells, wherein the first strand is processing the nucleic acid by RISC. Includes modified or unmodified nucleotides at multiple positions to facilitate.

In one aspect, "promoting processing by RISC" means that the nucleic acid can be processed by RISC, eg, by any modification present so that siRNA activity can occur appropriately. Means to make it possible.

Nucleotides at positions 2 and 14 from the 5'end of the first chain are not modified with 2'O-methyl modification and are at positions 11 or 13 or 11 and 13 or 11 of the first chain. Nucleotides (s) on the second strand corresponding to positions 12 and 13 are not modified with the 2'-OMe modification (in other words, they are not modified or with modifications other than 2'-OMe. (Modified), nucleic acids as disclosed herein.

In one aspect, the nucleotide on the second strand that corresponds to position 13 of the first strand is a nucleotide that base pairs with position 13 of the first strand.

In one aspect, the nucleotide on the second strand that corresponds to the 11th position of the first strand is a nucleotide that base pairs with the 11th position of the first strand.

In one aspect, the nucleotide on the second strand that corresponds to the 12th position of the first strand is the nucleotide that forms a base pair with the 12th position of the first strand.

For example, in a double-stranded and blunt-ended 19-mer nucleic acid, the 13-position of the first strand pairs with the 7-position of the second strand. The 11th position of the first chain is paired with the 9th position of the second chain. This system can be applied to other positions in the second strand.

In one aspect, in the case of the partially complementary first and second strands, the nucleotides on the second strand that "correspond" to the position on the first strand have a mismatch in that position. However, if the principle of the system is still applicable, it does not necessarily have to form a base pair.

In one embodiment, the nucleotides at positions 2 and 14 from the 5'end of the first strand are not modified with the 2'-OMe modification and are at positions 11 or 13 or 11 and 13 of the first strand. Nucleotides on the second strand corresponding to positions, or 11th to 13th positions, are nucleic acids as disclosed herein that are not modified with the 2'-F modification.

In one embodiment, the nucleotides at positions 2 and 14 from the 5'end of the first strand are modified with 2'-F modification and are at positions 11 or 13 or 11 and 13 of the first strand. , Or the nucleotides on the second strand corresponding to positions 11-13 are nucleic acids as disclosed herein that are modified with a 2'-OMe modification.

In one embodiment, the nucleotides at positions 2 and 14 from the 5'end of the first strand are modified with 2'-F modification and are at positions 11 or 13 or 11 and 13 of the first strand. , Or the nucleotides on the second strand corresponding to positions 11-13 are nucleic acids as disclosed herein that are modified with a 2'-F modification.

In one embodiment, more than 50% of the nucleotides in the first and / or second strand, preferably measured as a percentage of the total nucleotides in both the first and second strands, comprises the 2'-OMe modification. For example, 55%, 60%, 65%, 70%, 75%, 80%, or 85%, or more, the first and / or second strands comprising a 2'-OMe modification. Nucleic acids as disclosed in the book.

One embodiment comprises a naturally occurring RNA modification in which more than 50% of the first and / or second strands of nucleotides, preferably measured as a percentage of the total nucleotides of both the first and second strands. , For example, 55%, 60%, 65%, 70%, 75%, 80%, or 85%, or more, the first and / or second strands comprising such modifications herein. It is a nucleic acid as disclosed in. Suitable naturally occurring modifications include not only 2'-OMe, but also other 2'sugar modifications, in particular 2'-H modifications that result in DNA nucleotides.

One embodiment preferably has a 2'modification that is not a 2'-OM modification on the first and / or second strand, preferably as a percentage of total nucleotides in both the first and second strands, 20% or less, Nucleic acids as disclosed herein, comprising, for example, 15% or less, eg, 10% or less, nucleotides.

In one embodiment, the number of nucleotides in the first and / or second strand, having a 2'modification that is not a 2'-OMe modification, is 7 or less, more preferably 5 or less, most preferably 3 or less. Certain nucleic acids as disclosed herein.

One embodiment is preferably 20% or less (eg, 15% or less or 10%) of the 2'-F modification on the first and / or second strand, preferably as a percentage of total nucleotides in both the first and second strands. Nucleic acids as disclosed herein, including:

In one embodiment, the number of nucleotides in the first and / or second strand with the 2'-F modification is 7 or less, more preferably 5 or less, most preferably 3 or less, herein. Nucleic acids as disclosed therein.

One aspect corresponds to the 2nd and 14th positions from the 5'end of the first chain, and the 11th or 13th position, or the 11th and 13th positions, or the 11th to 13th positions of the first chain. All nucleotides, with the exception of nucleotides on the strand of, are nucleic acids as disclosed herein, modified with a 2'-OMe modification. Preferably, nucleotides that are not modified with 2'-OMe are modified with fluoro at the 2'position (2'-F modification).

Nucleic acids as disclosed herein are preferred, in which all nucleotides of the nucleic acid are modified at the 2'position of the sugar. Preferably, these nucleotides are modified with a 2'-F modification, where the modification is not a 2'-OMe modification.

In one aspect, the nucleic acid is modified on the first strand with alternating 2'-OMe and 2-F modifications, with positions 2 and 14 (starting at the 5'end) at 2'-F. Be qualified. Preferably, the second strand is 2'-F at the nucleotides on the second strand corresponding to the 11th, 13th, or 11th and 13th positions, or 11th to 13th positions of the first strand. It is modified by modification. Preferably, the second strand is modified with a 2'-F modification at positions 11-13 counting from the 3'end starting at the first position of the complementary (double chain) region and the remaining modifications. Is a naturally occurring modification, preferably 2'-OMe.

In one aspect of nucleic acid, the nucleotides in the first and second strands are modified nucleotides, respectively.

The term "odd numbered" as used herein means a number that is not divisible by two. Examples of odd numbers are 1, 3, 5, 7, 9, 11 and so on. One or more of the even numbered nucleotides of the first strand of the nucleic acid of the invention may be modified, where the first strand is numbered from 5'to 3'. To. The term "numbered even", as used herein, means a number evenly divisible by two. Examples of even numbers are 2, 4, 6, 8, 10, 12, 14, and so on.

As used herein, nucleotides in the first strand are numbered sequentially starting with nucleotide number 1 at the 5'end of the first strand. Nucleotides in the second strand are numbered sequentially starting with nucleotide number 1 at the 3'end of the second strand.

One or more nucleotides on the first and / or second strand can be modified to form modified nucleotides. One or more of the odd numbered nucleotides in the first strand may be modified. One or more of the even numbered nucleotides in the first strand may be modified by at least the second modification, where at least the second modification is to one or more odd nucleotides. Different from modification. At least one of the one or more modified even numbered nucleotides may be adjacent to at least one of the one or more modified odd numbered nucleotides.

The plurality of odd numbered nucleotides in the first strand may be modified in the nucleic acids of the invention. The plurality of even numbered nucleotides in the first strand may be modified with the second modification. The first strand may contain adjacent nucleotides that are modified by a common modification. The first strand may also contain adjacent nucleotides modified by a second different modification (ie, the first strands are adjacent to each other and the nucleotides modified by the first modification as well as adjacent to each other. And may include other nucleotides modified by a second modification that is different from the first modification).

One or more of the odd numbered nucleotides in the second strand, where the nucleotides are numbered consecutively starting with nucleotide number 1 at the 3'end of the second strand. Modifications by modifications different from those of the odd numbered nucleotides of the first strand, where the nucleotides are numbered sequentially starting with nucleotide number 1 at the 5'end of the first strand. And / or one or more of the nucleotides numbered even in the second strand may be modified by the same modification of the nucleotides numbered odd in the first strand. At least one of the one or more modified even numbered nucleotides in the second strand may be adjacent to one or more modified odd numbered nucleotides. Multiple odd numbered nucleotides in the second strand may be modified by a common modification and / or multiple even numbered nucleotides are numbered odd numbered in the first strand. It may be modified by the same modification present on the nucleotide. The odd numbered nucleotides on the second strand may be modified by modifications different from those of the odd numbered nucleotides on the first strand.

The second strand may contain adjacent nucleotides modified by a common modification, which may be a modification different from the modification of the odd numbered nucleotides of the first strand.

In the nucleic acids of the invention, the odd numbered nucleotides in the first strand and the even numbered nucleotides in the second strand may each be modified by a common modification and are even numbered. Each numbered nucleotide may be modified with a different modification in the first strand, and each odd numbered nucleotide may be modified with a different modification in the second strand.

The nucleic acids of the invention may have first strand modified nucleotides shifted by at least one nucleotide relative to a second strand unmodified nucleotide or a differently modified nucleotide.

One or more of the odd numbered nucleotides, or each, may be modified in the first strand, and one or more of the even numbered nucleotides, or each, in the second strand. It may be modified. One or more of the alternating nucleotides on one or both strands, or each, may be modified by a second modification. One or more of the even numbered nucleotides, or each, may be modified in the first strand, and one or more of the even numbered nucleotides, or each in the second strand. It may be modified. One or more of the alternating nucleotides on one or both strands, or each, may be modified by a second modification. One or more of the odd numbered nucleotides, or each, may be modified in the first strand, and one or more of the odd numbered nucleotides, or each, in the second strand. It may be modified by a common modification. One or more of the alternating nucleotides on one or both strands, or each, may be modified by a second modification. One or more of the even numbered nucleotides, or each, may be modified in the first strand, and one or more of the odd numbered nucleotides, or each in the second strand. It may be modified by a common modification. One or more of the alternating nucleotides on one or both strands, or each, may be modified by a second modification.

The nucleic acids of the invention may comprise a single or double chain construct containing at least two regions of alternating modification in one or both of the strands. These alternating regions may contain up to about 12 nucleotides, but preferably about 3 to about 10 nucleotides. Regions of alternating nucleotides can be located at the ends of one or both strands of the nucleic acids of the invention. The nucleic acid may contain 4 to about 10 nucleotides of alternating nucleotides at each terminal (3'and 5'), these regions containing about 5 to about 12 adjacent unmodified nucleotides. Or they can be separated with different or commonly modified nucleotides.

The odd numbered nucleotides of the first strand may be modified and the even numbered nucleotides may be modified with the second modification. The second strand may contain adjacent nucleotides modified with a common modification, which may be the same as the modification of the odd numbered nucleotides of the first strand. One or more nucleotides in the second strand may also be modified with the second modification. One or more nucleotides with the second modification may be adjacent to each other and to nucleotides with modifications that are the same as those of the odd numbered nucleotides of the first strand. The first strand may also contain a phosphorothioate bond between the two nucleotides at the 3'and 5'ends. The second strand may contain a phosphorothioate bond between the two nucleotides at the 5'end. The second strand may also be conjugated to a ligand at the 5'end.

The nucleic acids of the invention may comprise a first strand containing adjacent nucleotides modified with a common modification. One or more such nucleotides may be flanked by one or more nucleotides that may be modified with the second modification. One or more nucleotides with the second modification may be flanked. The second strand may contain adjacent nucleotides modified with a common modification, which may be the same as one of the modifications of one or more nucleotides of the first strand. One or more nucleotides in the second strand can also be modified with the second modification. One or more nucleotides with the second modification may be flanked. The first strand may also contain a phosphorothioate bond between the two nucleotides at the 5'and 3'ends. The second strand may contain a phosolothioate bond between the two nucleotides at the 3'end. The second strand may also be conjugated to a ligand at the 5'end.

From 5'to 3'on the first chain and from 3'to 5'on the second chain 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25 The numbered nucleotides can be modified by modification on the first strand. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 can be modified by a second modification on the first strand. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 can be modified by modification on the second strand. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 can be modified by a second modification on the second strand. Nucleotides are numbered 5'to 3'on the first strand and 3'to 5'on the second strand for the nucleic acids of the invention.

Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 can be modified by modification on the first strand. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 can be modified by a second modification on the first strand. Nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 can be modified by modification on the second strand. Nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 can be modified by a second modification on the second strand.

Obviously, if the first and / or second strands are shorter than 25 nucleotides in length, eg 19 nucleotides in length, they are numbered 20, 21, 22, 23, 24 and 25 to be modified. Nucleotides are absent. Therefore, it will be appreciated by those skilled in the art to apply the above description to shorter chains.

One or more modified nucleotides on the first strand may be paired with modified nucleotides on the second strand having a common modification. One or more modified nucleotides on the first strand may be paired with modified nucleotides on the second strand with different modifications. One or more modified nucleotides on the first strand may be paired with unmodified nucleotides on the second strand. One or more modified nucleotides on the second strand may be paired with unmodified nucleotides on the first strand. In other words, for example, all modifications in the alternating regions of the second strand are paired with the same modification in the first strand, or the modification is a different modification in the other strand (ie, the second strand). The alternating nucleotides should be aligned on the two strands so that they can be offset by one nucleotide with a common modification in the alternating region of one strand paired with the modification or further modification). Can be done. Another option is to have different modifications in each chain.

Modifications on the first strand may be shifted by one nucleotide with respect to the modified nucleotides on the second strand so that the common modified nucleotides are not paired with each other.

Modifications (single and / or plural) are individually 3'terminal deoxythymine, 2'-OMe, 2'deoxy modification, 2'amino modification, 2'alkyl modification, morpholino modification, phosphoramidate modification, 5'. -It may be selected from the group consisting of phosphorothioate group modifications, 5'phosphate or 5'phosphate mimetic modifications and cholesteryl derivatives or dodecanoic acid bisdecylamide group modifications, and / or the modified nucleotides may be locked nucleic acids, debase nucleotides or It may be any one of the nucleotides containing an unnatural base.

At least one modification may be 2'-OMe and / or at least one modification may be 2'-F. Further modifications as described herein may be present on the first and / or second strand.

The nucleic acids of the invention may contain reverse RNA nucleotides at one or several ends of the strand. Such reverse nucleotides provide stability to the nucleic acid. Preferably, the nucleic acid comprises at least a reverse nucleotide at the 3'end of the first and / or second strand and / or at the 5'end of the second strand. More preferably, the nucleic acid comprises a reverse nucleotide at the 3'end of the second strand. Most preferably, the nucleic acid comprises a reverse RNA nucleotide at the 3'end of the second strand, which nucleotide is preferably reverse A. Reverse nucleotides may be linked to the 3'end of the nucleic acid by the 3'carbon, not by its 5'carbon, which would normally be the case, or by the 3'carbon, which would normally be the case. It is a nucleotide linked to the 5'end of nucleic acid by its 5'carbon. The reverse nucleotide is preferably present at the end of the strand, opposite the corresponding nucleotide in the other strand, rather than as an overhang. Therefore, nucleic acids are preferably blunt-ended at the ends containing reverse RNA nucleotides. The presence of a reverse RNA nucleotide at the end of a strand preferably means that the last nucleotide at this end of the strand is a reverse RNA nucleotide. Nucleic acids having such nucleotides are stable and easy to synthesize. The reverse RNA nucleotide is preferably an unmodified nucleotide in the sense that it does not contain any modifications as compared to its native nucleotide counterpart. Specifically, the reverse RNA nucleotide is preferably a 2'-OH nucleotide.

The nucleic acid of the invention may comprise one or more nucleotides modified with 2'-H at the 2'position and thus having DNA nucleotides within the nucleic acid. The nucleic acid of the invention may contain DNA nucleotides at positions 2 and / or 14 of the first strand, counting from the 5'end of the first strand. The nucleic acid may contain a DNA nucleotide on the second strand corresponding to the 11th, or 13th, or 11th and 13th, or 11th to 13th positions of the first strand.

In one aspect, there are no more than one DNA nucleotide per nucleic acid of the invention.

The nucleic acid of the present invention may contain one or more LNA nucleotides. The nucleic acid of the present invention may contain LNA nucleotides at positions 2 and / or 14 of the first strand, counting from the 5'end of the first strand. The nucleic acid may contain LNA on the second strand corresponding to the 11th or 13th position, or the 11th and 13th positions, or the 11th to 13th positions of the first strand.

Some representative modified nucleic acid sequences of the invention are shown in Examples. These examples are intended to be representative and non-limiting.

Preferably, the nucleic acids may individually comprise a modification selected from the group comprising a 2'-OMe modification and a 2'-F modification and a second modification or further modification. The nucleic acid may include a modification that is 2'-OMe, which may be the first modification, and a second modification that is 2'-F. The nucleic acids of the invention may also have phosphorothioate modifications and / or modifications that may be present at or between the two or three nucleotides at the ends of each or both ends of each or both strands. Deoxy modifications may be included.

In one aspect of nucleic acid, at least one nucleotide in the first and / or second strand is a modified nucleotide, where the first strand contains at least one modified nucleotide.
(i) At least one or both of nucleotides 2 and 14 of the first strand are modified by the first modification and / or
(ii) At least one, some, or all of the even numbered nucleotides of the first strand are modified by the first modification and / or
(iii) At least one, some, or all of the odd numbered nucleotides of the first strand are modified by the second modification, and / or where the second strand is at least one. If it contains modified nucleotides of
(iv) At least one, some, or all of the nucleotides of the second strand at positions corresponding to the even numbered nucleotides of the first strand are modified by the third modification and / or
(v) At least one, some, or all of the nucleotides of the second strand at positions corresponding to the odd numbered nucleotides of the first strand are modified by the fourth modification and / or
(vi) At least one, some, or all of the nucleotides of the second strand at positions corresponding to nucleotides 11 or 13 of the first strand or nucleotides 11 and 13 or nucleotides 11-13 are the fourth modification. Modified by and / or
(vii) At least one, some, or all of the nucleotides of the second strand at positions other than nucleotides 11 or 13 of the first strand or positions corresponding to nucleotides 11 and 13 or nucleotides 11-13. Qualified by 3 modifications,
Here, the nucleotides on the first strand are numbered sequentially starting with nucleotide number 1 at the 5'end of the first strand.
The modification is preferably at least one of the following:
(a) The first modification is preferably different from the second modification and the third modification.
(b) The first modification is preferably the same as the fourth modification,
(c) The second and third modifications are preferably the same modifications.
(d) The first modification is preferably a 2'-F modification,
(e) The second modification is preferably a 2'-OMe modification.
(f) The third modification is preferably a 2'-OMe modification and / or
(g) The fourth modification is preferably a 2'-F modification,
Here, optionally, the nucleic acid is conjugated to the ligand.

One embodiment is a double-stranded nucleic acid, preferably for inhibiting expression of C3 in cells, wherein the nucleic acid comprises a first strand and a second strand, wherein the sequence of the first strand is , SEQ ID NOs: 361, 95, 111, 125, 131, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 , 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 , 89, 91, 93, 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 127, 129 or 133, preferably SEQ ID NOs: 361, 95, 111, 125. Or contains a sequence of at least 15 nucleotides in which any one of the 131 sequences and 3 or less nucleotides are different, where all the even numbered nucleotides in the first strand are by the first modification. All of the modified and odd numbered nucleotides of the first strand are modified by the second modification and all of the second strand nucleotides at positions corresponding to the even numbered nucleotides of the first strand. Is modified by the third modification, and all nucleotides in the second strand at positions corresponding to the odd numbered nucleotides in the first strand are modified by the fourth modification, where the first and the first and the other are. The fourth modification is 2'-F and the second and third modifications are 2'-OMe.

One embodiment is a double-stranded nucleic acid, preferably for inhibiting expression of C3 in cells, wherein the nucleic acid comprises a first strand and a second strand, wherein the sequence of the first strand is , SEQ ID NOs: 361, 95, 111, 125, 131, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 , 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 , 89, 91, 93, 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 127, 129 or 133, preferably SEQ ID NOs: 361, 95, 111, 125. Or contains a sequence of at least 15 nucleotides in which any one of the 131 sequences and 3 or less nucleotides are different, where all the even numbered nucleotides in the first strand are by the first modification. All of the modified and odd numbered nucleotides of the first strand are modified by the second modification, and all of the nucleotides of the second strand at positions corresponding to nucleotides 11 to 13 of the first strand are second. All the nucleotides of the second strand except the nucleotides corresponding to the nucleotides 11 to 13 of the first strand, which are modified by the modification of 4, are modified by the third modification, where the first and fourth modifications are made. , 2'-F, and the second and third modifications are 2'-OMe.

The 3'and 5'ends of oligonucleotides can be modified. Such modifications may be present at the 3'and / or 5'ends of the molecule. Such modifications may include modification or replacement of the entire terminal phosphate or one or more atoms of the phosphate group. For example, the 3'and 5'ends of oligonucleotides are labeled moieties such as fluorophores (eg pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (eg based on sulfur, silicon, boron or esters) and the like. It can be conjugated to other functional molecular entities. The functional molecular entity can be attached to the sugar by a phosphate group and / or a linker. The terminal atom of the linker can be attached to or replace the O, N, S or C group of the linking atom of the phosphate group or the C-3'or C-5' of the sugar. Alternatively, the linker may be attached to or replace the terminal atom of a nucleotide substitute (eg, PNA). These spacers or linkers include, for example,-(CH2) n-,-(CH2) nN-,-(CH2) nO-,-(CH2) nS-,-(CH2CH2O) nCH2CH2O- (eg n = 3 or 6), debasic sugars, amides, carboxys, amines, oxyamines, oxyimines, thioethers, disulfides, thioureas, sulfonamides or morpholinos, or biotin and fluorescein reagents. The 3'end can be a -OH group.

Other examples of end modifications include dyes, inserts (eg, acridin), cross-linking agents (eg, solarene, mitomycin C), polyphyllin (TPPC4, texaphyrin, sapphirine), polycyclic aromatic hydrocarbons (eg, phenazine, etc.). Dihydrophenazine), artificial endonucleases, EDTA, lipophilic carriers (eg cholesterol, cholic acid, adamantanacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O (hexadecyl) glycerol, geranyloxyhexyl groups, hexa Decylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3- (oleyl) lithocolic acid, O3- (oleyl) cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (Eg Antennapedia Peptide, Tat Peptide), Alkylating Agent, Phosphate, Amino, Mercapto, PEG (eg PEG-40K), MPEG, [MPEG] 2, Polyamino, Alkyl, Substituent Alkyl, Radiolabeling Marker, Enzyme, Hapten (eg, biotin), transport / absorption promoters (eg, aspirin, vitamin E, folic acid), synthetic ribonucleases (eg, imidazole, bisimidazole, histamine, imidazole clusters, acylin-imidazole conjugates, Eu3 + tetraaza macrocyclic compounds) Complex).

Terminal modification may also be useful for monitoring distribution, in which case the group to be added may include a fluorophore, such as fluorescein or an Alexa dye. Terminal modifications may also be useful for enhancing uptake, including cholesterol as a useful modification. Terminal modification can also be useful for cross-linking the RNA agent to another moiety.

Terminal modification can be added for a number of reasons, including to regulate activity or resistance to degradation. Terminal modifications useful for regulating activity include modification with a phosphate or phosphate analog at the 5'end. The nucleic acids of the invention may be 5'phosphorylated on the first or second strand, or may contain a phosphoryl analog at the first end of the 5'. 5'-phosphate modifications include those that are compatible with RISC-mediated gene silencing. As appropriate modifications, 5'-monophosphate ((HO) ₂ (O) PO-5');5'-diphosphate ((HO) ₂ (O) POP (HO) (O) -O-5') 5'-Triphosphate ((HO) ₂ (O) PO- (HO) (O) POP (HO) (O) -O-5');5'-Ganosincap (7-methylated or methylated) Not) (7m-GO-5'-(HO) (O) PO- (HO) (O) POP (HO) (O) -O-5');5'-Adenosine cap (Appp), and optional Modified or unmodified nucleotide cap structure (NO-5'-(HO) (O) PO- (HO) (O) POP (HO) (O) -O-5');5'-monothiophosphate (phosphorothioate) (HO) ₂ (S) PO-5');5'-monodithiophosphate(phosphologithioate; (HO) (HS) (S) PO-5'), 5'-phosphorothiolate (((HO) 2 (S) PO-5'); HO) ₂ (O) PS-5'); Oxygen / sulfur replaced monophosphate, diphosphate and triphosphate (eg 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5 '-Phosphorumidate ((HO) ₂ (O) P-NH-5', (HO) (NH ₂ ) (O) PO-5'), 5'-alkylphosphonate (alkyl = methyl, ethyl, isopropyl) , Propyl, etc., for example, RP (OH) (O) -O-5'-(in the formula, R is alkyl), (OH) ₂ (O) P-5'-CH _2- ), 5'vinyl. Phosphonate, 5'-alkyl ether phosphonate (alkyl ether = methoxymethyl (MeOCH _2- ), ethoxymethyl, etc., eg RP (OH) (O) -O-5'-(in the formula, R is an alkyl ether). Any further combination of).

Certain portions may be linked to the 5'end of the first or second strand. These include a debasement ribose moiety, a debasement deoxyribose moiety, a modified debasement ribose and a debasement deoxyribose moiety including a 2'-O alkyl modification; a reverse debasement ribose and a debasement deoxyribose moiety and their modifications, C6. -Imino-Pi; Mirror nucleotides containing L-DNA and L-RNA; 5'OMe nucleotides; and nucleotide analogs containing 4', 5'-methylene nucleotides; 1- (β-D-erythrofuranosyl) nucleotides; 4'-thionucleotide, carbon cyclic nucleotide; 5'-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate 1,5-Anhydrohexitol nucleotides; Alpha-nucleotides; Threo-pentoflanosyl nucleotides; Acyclic 3', 4'-seconucleotides; 3,4-dihydroxybutyl nucleotides; 3,5-Dihydroxypentylnucleotides, 5'-5'-reverse debasement moieties; 1,4-butanediol phosphate; 5-amino; as well as crosslinkable or non-bridged methylphosphonates and 5'-mercapto moieties.

The invention also provides nucleic acids according to any aspect of the invention described herein, wherein the first strand has a terminal 5'(E) -vinylphosphonate nucleotide at its 5'end. Have. This terminal 5'(E) -vinylphosphonate nucleotide is preferably linked to the second nucleotide in the first strand by a phosphodiester bond.

The first strand of nucleic acid may contain formula (I):
(vp)-N _(po) [N _(po) ] _n- (I)
(In the formula, "(Vp)-" is 5'(E) -vinyl phosphonate, "N" is a nucleotide, "po" is a phosphodiester bond, and n is 1 to (No. 1). The total number of nucleotides in one strand-2), preferably n is 1 to (total number of nucleotides in the first strand-3), and more preferably n is 1 to (total number of nucleotides in the first strand-3). The total number of nucleotides in -4)).

Preferably, the terminal 5'(E) -vinylphosphonate nucleotide is an RNA nucleotide, preferably (vp) -U.

The terminal 5'(E) -vinylphosphonate nucleotide is a nucleotide in which the natural phosphate group at the 5'end is replaced with E-vinylphosphonate, where the crosslinkability of the terminal nucleotide of the 5'phosphorylated chain is 5'. -Oxygen atoms are replaced with methinyl (-CH =) groups:

5'(E) -vinyl phosphonate is a 5'phosphate mimetic. A biological mimetic is a molecule that is capable of performing the same function as the original molecule being mimicked and is structurally very similar to the original molecule being mimicked. In the context of the present invention, the 5'(E) -vinyl phosphonate mimetic mimics the function of normal 5'phosphate, allowing, for example, efficient RISC loading. In addition, due to the slightly modified structure, 5'(E) -vinyl phosphonate can stabilize the 5'terminal nucleotide by protecting it from dephosphorylation by enzymes such as phosphatase. be.

In one aspect, the first strand has a terminal 5'(E) -vinylphosphonate nucleotide at its 5'end and the terminal 5'(E) -vinylphosphonate nucleotide is the second nucleotide in the first strand. The first strand is a) more than one phosphodiester bond, b) a phosphodiester bond between at least the three 5'nucleotides at the end and / or c) at least the four at the end. Contains phosphodiester bonds between 5'nucleotides.

In one aspect, the first and / or second strand of nucleic acid comprises at least one phosphorothioate (ps) bond between two nucleotides.

In one aspect, the first and / or second strand of nucleic acid comprises more than one phosphorothioate bond.

In one aspect, the first and / or second strand of the nucleic acid comprises a phosphorothioate bond between the two terminal 3'nucleotides or a phosphorothioate bond between the terminal three 3'nucleotides. Preferably, the bond between the other nucleotides in the first and / or second strand is a phosphodiester bond.

In one aspect, the first and / or second strand of the nucleic acid comprises a phosphorothioate bond between the two terminal 5'nucleotides or a phosphorothioate bond between the three terminal 5'nucleotides.

In one aspect, the nucleic acids of the invention comprise one or more phosphorothioate modifications on one or more of the ends of the first and second strands. Optionally, each or one end of the first strand may contain one or two or three phosphorothioate-modified nucleotides. Optionally, each or one end of the second strand may contain one or two or three phosphorothioate-modified nucleotides.

In one aspect, the nucleic acid comprises a phosphorothioate bond between two or three 3'nucleotides and / or 5'nucleotides at the ends of the first and / or second strand. Preferably, the nucleic acid comprises a phosphorothioate bond between each of the three 3'nucleotides at the end of the first and / or second strand and the three 5'nucleotides at the end. Preferably, all remaining bonds between the nucleotides of the first and / second strands are phosphodiester bonds.

In one embodiment, the nucleic acid comprises a phosphorodithioate bond between each of the 2, 3 or 4 terminal nucleotides at the 3'end of the first strand, and / or 3 of the second strand. 'A phosphorodithioate bond between each of the two, three or four terminal nucleotides at the end, and / or the two, three or four ends at the 5'end of the second strand. It contains a phosphorodithioate bond between each of the nucleotides and contains non-phosphologithioate bonds between the 2, 3 or 4 terminal nucleotides at the 5'end of the first strand.

In one aspect, the nucleic acid comprises a phosphorothioate bond between the three 3'nucleotides at the ends of the first and second strands and the three 5'nucleotides at the end. Preferably, all remaining bonds between the nucleotides of the first strand and / or the second strand are phosphodiester bonds.

In one aspect, the nucleic acid is:
(i) It has a phosphorothioate bond between the three 3'nucleotides at the end of the first strand and the three 5'nucleotides at the end.
(ii) conjugated to a tri-branched ligand on either the 3'end nucleotide or the 5'end nucleotide of the second strand.
(iii) Having a phosphorothioate bond between the three nucleotides at the end of the second strand, at the end opposite to the end conjugated to the tribranched ligand.
(iv) All remaining bonds between the nucleotides of the first and / or second strand are phosphodiester bonds.

In one aspect, the nucleic acid is:
(i) It has a terminal 5'(E) -vinylphosphonate nucleotide at the 5'end of the first strand.
(ii) Have phosphorothioate bonds between the three 3'nucleotides at the ends above the first and second strands and between the three 5'nucleotides at the ends above the second strand.
(iii) All remaining bonds between the nucleotides of the first and / or second strand are phosphodiester bonds.

The use of phosphorodithioates in the nucleic acids of the invention reduces stereochemical variability in the population of nucleic acid molecules as compared to molecules containing phosphorothioates at their co-positions. Phosphorothioate binding introduces a chiral center and it is difficult to control which unconnected oxygen is replaced with sulfur. The use of phosphorodithioates ensures that the chiral center is not present in the binding, and therefore any variation in the population of nucleic acid molecules, depending on the number of phosphorodithioates and phosphorothioate bonds used in the nucleic acid molecule. To reduce or eliminate.

In one embodiment, the nucleic acid has a phosphologithioate bond between the two terminal nucleotides at the 3'end of the first strand and a phospho between the two terminal nucleotides at the 3'end of the second strand. Logithioate binding, also containing phosphorodithioate binding between the two terminal nucleotides at the 5'end of the second strand, 2, 3 or 4 at the 5'end of the first strand Contains bonds other than phosphorodithioate bonds between the terminal nucleotides of. Preferably, the first strand has a terminal 5'(E) -vinyl phosphonate at its 5'end. This terminal 5'(E) -vinylphosphonate nucleotide is preferably linked to the second nucleotide in the first strand by a phosphodiester bond. Preferably, for both strands other than the bond between the two terminal nucleotides at the 3'end of the first strand and the bond between the two terminal nucleotides at the 3'and 5'ends of the second strand. All bonds between nucleotides are phosphodiester bonds.

In one embodiment, the nucleic acid is between each of the three terminal 3'nucleotides on the first strand and / or between each of the three terminal 5'nucleotides and / or the three of the second strand. Includes a phosphorothioate bond between each of the terminal 3'nucleotides and / or between each of the three terminal 5'nucleotides if no phosphorodithioate bond is present at the end. The absence of a phosphorodithioate bond at the end means that the bond between the two terminal nucleotides, or preferably between the three terminal nucleotides at the nucleic acid end in question, is a bond other than the phosphorodithioate bond. It means that there is.

In one embodiment, both strands other than the bond between the two terminal nucleotides at the 3'end of the first strand and the bond between the two terminal nucleotides at the 3'and 5'ends of the second strand. All nucleic acid bonds between nucleotides are phosphodiester bonds.

Other phosphate binding modifications are possible.

Phosphate linkers can also be modified by replacement of linked oxygen with nitrogen (crosslinked phosphoramidate), sulfur (crosslinked phosphorothioate) and carbon (crosslinked methylenephosphonate). The replacement can be done with terminal oxygen. It is possible to replace unconnected oxygen with nitrogen.

Phosphate groups can also be individually replaced with non-phosphorus-containing connectors.

Examples of moieties that can replace phosphate groups are siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thieoform acetal, form acetal, oxime, methylene imino, methylene methyl imino, Methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino can be mentioned. In certain embodiments, the substitution may include a methylenecarbonylamino and a methylenemethylimino group.

Phosphate linkers and ribose sugars can be replaced with nuclease-resistant nucleotides. Examples include morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside substitutes. In certain embodiments, PNA alternatives may be used.

In one aspect, the nucleic acid, preferably siRNA that inhibits the expression of complement component C3 by RNAi, comprises one or more or all of the following:
(i) Modified nucleotides;
(ii) Modified nucleotides other than the 2'-OMe modified nucleotides located at the 2nd and 14th positions from the 5'end of the first strand, preferably 2'-F modified nucleotides;
(iii) Each of the odd numbered nucleotides in the first strand, if numbered starting from 1 at the 5'end of the first strand, is a 2'-OMe modified nucleotide;
(iv) Each even numbered nucleotide in the first strand, if numbered starting from 1 at the 5'end of the first strand, is a 2'-F modified nucleotide;
(v) The nucleotides of the second strand corresponding to the 11th or 13th position of the first strand are modified by modifications other than the 2'-OMe modification, preferably one or both of these positions are 2'. -Includes F modification;
(vi) Reverse nucleotide at the 3'end of the second strand, preferably a 3'-3'bond;
(vii) One or more phosphorothioate bonds;
(viii) One or more phosphorodithioate linkages; and / or
(ix) The first strand has a terminal 5'(E) -vinylphosphonate nucleotide at its 5'end, where the terminal 5'(E) -vinylphosphonate nucleotide is preferably uridine, the first. It is preferably linked to the second nucleotide in the chain by a phosphodiester bond.

All nucleic acid features can be combined with all other aspects of the invention disclosed herein.

Ligand The nucleic acid of the invention can be conjugated to a ligand. Efficient delivery of the oligonucleotides of the invention, in particular double-stranded nucleic acids, to cells in vivo is important and requires substantial protection from specific targeting and extracellular environment, especially from serum proteins. .. One way to achieve specific targeting is to conjugate the ligand to the nucleic acid. The ligand helps target the nucleic acid to the desired target site. In order for the conjugated molecule to be taken up by the target cell by a mechanism such as various receptor-mediated endocytosis pathways or functionally similar processes, it is conjugated with a ligand suitable for the desired receptor molecule. There is a need.

One example is the asialoglycoprotein receptor complex (ASGP-R), which is composed of multimers of various ratios of membrane ASGR1 and ASGR2 receptors, which are highly abundant in hepatocytes and also herein. It has a high affinity for the GalNAc moiety described in the book. One of the first disclosures of the use of tri-branched cluster glycosides as conjugated ligands was US Pat. No. 5,885,968. A conjugate having three GalNAc ligands and containing a phosphate group is known and described in Dubber et al. (Bioconjug. Chem. 2003 Jan-Feb; 14 (1): pp. 239-46). The ASGP-R complex shows a 50-fold higher affinity for N-acetyl-D-galactosamine (GalNAc) than D-Gal.

Asialoglycoprotein receptor complex (ASGP-R) (Weigel, PH et al., Biochim. Biophys. Acta. 2002) that specifically recognizes the terminal β-galactosyl subunit of glycosylated proteins or other oligosaccharides. May 19; 1572 (2-3): pp. 341-63) to deliver the drug to liver hepatocytes expressing the receptor complex by co-binding of galactose or galactosamine to the drug substance. Can be used (Ishibashi, S. et al., J Biol. Chem. November 11, 1994; 269 (45): 27803-6). In addition, binding affinity can be significantly increased by the effect of polyvalence achieved by iteration of the targeting molecule (Biessen EA et al., J Med Chem. 28 April 1995; 38 (9): 1538-46). ).

The ASGP-R complex is a mediator for the active uptake of terminal β-galactosyl-containing glycoproteins into the endosomes of cells. Therefore, ASGPR is well suited for targeted delivery of drug candidates conjugated to such ligands, such as nucleic acids, to receptor-expressing cells (Akinc et al., Mol Ther. July 2010; 18 (7): 1357-64).

More generally, the ligand may include a saccharide selected to have an affinity for at least one type of receptor on the target cell. In particular, the receptor is present on the surface of mammalian hepatocytes and is, for example, the aforementioned hepatic asialoglycoprotein receptor complex (ASGP-R).

The sugar can be selected from N-acetylgalactosamine, mannose, galactose, glucose, glucosamine and fucose. The saccharide may be N-acetylgalactosamine (GalNAc).

Thus, a ligand for use in the present invention is such that (i) one or more N-acetylgalactosamine (GalNAc) moieties and derivatives thereof, and (ii) GalNAc moieties are defined in any of the above embodiments. It may contain a linker that conjugates to a different nucleic acid. The linker can be a monovalent structure or a divalent or trivalent or tetravalent branched structure. Nucleotides can be modified as defined herein.

Therefore, the ligand may include GalNAc.

In one aspect, the nucleic acid is expressed in formula (II) :.
[SX ¹ -PX ² ] ₃ -AX ^3- (II)
(During the ceremony,
S represents a saccharide, preferably the saccharide is N-acetylgalactosamine.
X ¹ represents C ₃ to C ₆ alkylene or (-CH ₂ -CH ₂ -O) _m (-CH ₂ ) _2- (in the formula, m is 1, 2 or 3).
P is phosphate or modified phosphate, preferably thiophosphate, and
X ² is an alkylene or an alkylene ether of the formula (-CH ₂ ) _n -O-CH _2- (in the formula, n = 1 to 6).
A is a branch unit,
X ³ represents the crosslinkable unit
Here, the nucleic acid according to the invention is conjugated to X ³ by phosphate or modified phosphate, preferably thiophosphate).
It is conjugated to a ligand containing the compound of.

In formula (II), the branching unit "A" is preferably branched into three to accommodate the three saccharide ligands. The branching unit is preferably covalently attached to the remaining tether moiety of the ligand and the nucleic acid. The branching unit may include a branched aliphatic group containing a group selected from alkyl, amide, disulfide, polyethylene glycol, ether, thioether and hydroxyamino groups. The branching unit may include a group selected from alkyl and ether groups.

Branch unit A is

(In the equation, A ₁ independently represents O, S, C = O or NH, and n independently represents an integer from 1 to 20).
Can have a structure selected from.

The branch unit is

(In the equation, A ₁ independently represents O, S, C = O or NH, and n independently represents an integer from 1 to 20).
Can have a structure selected from.

The branch unit is

(In the equation, A ₁ is O, S, C = O or NH, and n independently represents an integer from 1 to 20)
Can have a structure selected from.

The branch unit is the structure:

May have.

The branch unit is the structure:

May have.

The branch unit is the structure:

May have.

Alternatively, the branch unit A is

(During the ceremony,
R ¹ is hydrogen or C ₁ to C ₁₀ alkylene,
R ² is C ₁ to C ₁₀ alkylene)
Can have a structure selected from.

Optionally, the branching unit consists of only carbon atoms.

The "X ³ " part is a crosslinkable unit. The crosslinkable unit is linear and is covalently attached to the branching unit and nucleic acid.

X ³ is -C ₁ to C ₂₀ alkylene-, -C ₂ to C ₂₀ alkenylene-, formula-(C ₁ to C ₂₀ alkylene) -O- (C ₁ to C ₂₀ alkylene) -alkylene ether, -C. (O) -C ₁ to C ₂₀ alkylene-, -C ₀ to C ₄ alkylene (Cy) C ₀ to C ₄ alkylene- (In the formula, Cy is a substituted or unsubstituted 5- or 6-membered cycloalkylene, arylene. , Heterocyclylene or heteroarylene ring), -C ₁ to C ₄ alkylene-NHC (O) -C ₁ to C ₄ alkylene-, -C ₁ to C ₄ alkylene-C (O) NH-C ₁ to C ₄ alkylene-, -C ₁ to C ₄ alkylene-SC (O) -C ₁ to C ₄ alkylene-, -C ₁ to C ₄ alkylene-C (O) SC ₁ to C ₄ alkylene-, -C ₁ to C ₄ alkylene-OC (O) -C ₁ to C ₄ alkylene-, -C ₁ to C ₄ alkylene-C (O) OC ₁ to C ₄ alkylene-, and C ₁ to C ₆ alkylene-SSC ₁ to C ₆ Can be selected from alkylene-.

X ³ may be an alkylene ether of the formula-(C ₁ to C ₂₀ alkylene) -O- (C ₁ to C ₂₀ alkylene)-. X ³ may be an alkylene ether of the formula-(C ₁ to C ₂₀ alkylene) -O- (C ₄ to C ₂₀ alkylene)-where the above (C ₄ to C ₂₀ alkylene) is Z. Is linked to. X ³ is -CH ₂ -OC ₃ H _6- , -CH ₂ -OC ₄ H _8- , -CH ₂ -OC ₆ H ₁₂ -and -CH ₂ -OC ₈ H _16- , especially -CH ₂ -OC It may be selected from the group consisting of ₄ H _8- , -CH ₂ -OC ₆ H _12- and -CH ₂ -OC ₈ H _16- , where in each case the -CH ₂ -group is Connected to A.

In one aspect, the nucleic acid is expressed in formula (III) :.
[SX ¹ -PX ² ] ₃ -AX ^3- (III)
(During the ceremony,
S stands for sugar, preferably GalNAc,
X ¹ represents C ₃ to C ₆ alkylene or (-CH ₂ -CH ₂ -O) _m (-CH ₂ ) _2- (in the formula, m is 1, 2 or 3).
P is phosphate or modified phosphate, preferably thiophosphate, and
X ² is C ₁ to C ₈ alkylene,
A is

It is a branch unit selected from
X ³ is a crosslinkable unit,
Here, the nucleic acid according to the invention is conjugated to X ³ by phosphate or modified phosphate, preferably thiophosphate).
It is conjugated to a ligand containing the compound of.

Branch unit A is the structure:

May have.

Branch unit A is the structure:

Where X ³ is attached to the nitrogen atom.

X ³ can be C ₁ to C ₂₀ alkylene. Preferably, X ³ is -C ₃ H _8- , -C ₄ H _8- , -C ₈ H _12- and -C ₈ H _16- , especially -C ₄ H _8- , -C ₈ H ₁₂ -and. -C ₈ H ₁₆ -Selected from the group consisting of-.

In one aspect, the nucleic acid is expressed in formula (IV) :.
[SX ¹ -PX ² ] ₃ -AX ^3- (IV)
(During the ceremony,
S stands for sugar, preferably GalNAc,
X ¹ represents C ₃ to C ₆ alkylene or (-CH ₂ -CH ₂ -O) _m (-CH ₂ ) _2- (in the formula, m is 1, 2 or 3).
P is phosphate or modified phosphate, preferably thiophosphate, and
X ² is an alkylene ether of the formula -C ₃ H ₆ -O-CH _2-
A is a branch unit,
X ³ is -CH ₂ -O-CH _2- , -CH ₂ -OC ₂ H _4- , -CH ₂ -OC ₃ H _6- , -CH ₂ -OC ₄ H _8- , -CH ₂ -OC ₅ An alkylene ether of the formula selected from the group consisting of H _10- , -CH ₂ -OC ₆ H _12- , -CH ₂ -OC ₇ H _14- , and -CH ₂ -OC ₈ H _16- , where In each case, the -CH ₂ -group is linked to A and X ³ is conjugated to the nucleic acid according to the invention by phosphate or modified phosphate, preferably thiophosphate).
It is conjugated to a ligand containing the compound of.

The branching unit may include carbon. Preferably, the branching unit is carbon.

X ³ is -CH ₂ -OC ₄ H _8- , -CH ₂ -OC ₅ H _10- , -CH ₂ -OC ₆ H _12- , -CH ₂ -OC ₇ H _14- , and -CH ₂ -OC It can be selected from the group consisting of ₈ H _16- . Preferably, X ³ is selected from the group consisting of -CH ₂ -OC ₄ H _8- , -CH ₂ -OC ₆ H _12- and -CH ₂ -OC ₈ H ₁₆ .

X ¹ can be (-CH ₂ -CH ₂ -O) (-CH ₂ ) _2- . X ¹ can be (-CH ₂ -CH ₂ -O) ₂ (-CH ₂ ) _2- . X ¹ can be (-CH ₂ -CH ₂ -O) ₃ (-CH ₂ ) _2- . Preferably, X ¹ is (-CH ₂ -CH ₂ -O) ₂ (-CH ₂ ) _2- . Alternatively, X ¹ represents C ₃ to C ₆ alkylene. X ¹ can be propylene. X ¹ can be butylene. X ¹ can be pentylene. X ¹ can be hexylene. Preferably, the alkyl is a linear alkylene. In particular, X ¹ can be butylene.

X ² represents the alkylene ether of the formula -C ₃ H ₆ -O-CH _2- , ie C ₃ alkoxymethylene, or -CH ₂ CH ₂ CH ₂ OCH _2- .

For any of the above embodiments, if P represents a modified phosphate group, then P is:

(In the equation, Y ¹ and Y ² are independent, = O, = S, ^-O- , -OH, -SH, -BH ₃ , -OCH ₂ CO ₂ , -OCH ₂ CO ₂ R ^x ,- Represents OCH ₂ C (S) OR ^x and -OR ^x (in the equation, R ^x represents C ₁ to C ₆ alkyl).

Indicates binding of the compound to the rest)
Can be represented by.

Modified phosphate means a phosphate group in which one or more of the unconnected oxygen has been replaced. Examples of modified phosphate groups include phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogenphosphonate, phosphoramidate, alkyl or arylphosphonate and phosphotriester. Phosphorodithioates have both unconnected oxygens replaced by sulfur. One, each or both unconnected oxygens in the phosphate group can be independently one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). ..

Phosphates can also be modified by replacement of linked oxygen with nitrogen (crosslinked phosphoramidate), sulfur (crosslinked phosphorothioate) and carbon (crosslinked methylenephosphonate). The replacement can be done with terminal oxygen. It is possible to replace unconnected oxygen with nitrogen.

For example, Y ¹ may represent -OH and Y ² may represent = O or = S, or
Y ¹ may represent -O ^- and Y ² may represent = O or = S.
Y ¹ may represent = O and Y ² may represent -CH ₃ , -SH, -OR ^x , or -BH ₃ .
Y ¹ may represent = S and Y ² may represent -CH ₃ , OR ^x or -SH.

It will be appreciated by those skilled in the art that delocalization is seen between Y ¹ and Y ² in certain cases.

Preferably, the modified phosphate group is a thiophosphate group. As thiophosphate groups, bithiophosphate (ie, where Y ¹ represents = S and Y ² represents ^-S- ) and monothiophosphate (ie, where Y ¹ represents -O). Represents Y ² for = S, or Y ¹ for = O and Y ² for ^-S- ). Preferably, P is monothiophosphate. We have found that conjugates with thiophosphate groups in the replacement of phosphate groups have improved potency and duration of action in vivo.

P may also be ethylphosphonate (ie, where Y ¹ represents = O and Y ² represents OCH ₂ CH ₃ ).

Sugars can be selected to have an affinity for at least one type of receptor on the target cell. In particular, the receptor is present on the surface of mammalian liver cells and is, for example, the hepatic asialoglycoprotein receptor complex (ASGP-R).

For any of the above or the following embodiments, the saccharide may be selected from N-acetyl having one or more of galactosamine, mannose, galactose, glucose, glucosamine and fructose. Generally, the ligand to be used in the present invention may include N-acetylgalactosamine (GalNAc). Preferably, the compounds of the invention may have three ligands, each of which preferably comprises N-acetylgalactosamine.

"GalNAc" refers to 2- (acetylamino) -2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetylgalactosamine. References to "GalNAc" or "N-acetylgalactosamine" include β-type: 2- (acetylamino) -2-deoxy-β-D-galactopyranose and α-type: 2- (acetylamino) -2-deoxy. -Includes both α-D-galactopyranose. In certain embodiments, β-type: 2- (acetylamino) -2-deoxy-β-D-galactopyranose and α-type: 2- (acetylamino) -2-deoxy-α-D-galacto Both pyranose can be used interchangeably. Preferably, the compounds of the invention include the β-type 2- (acetylamino) -2-deoxy-β-D-galactopyranose.

2- (Acetylamino) -2-deoxy-D-galactopyranose

2- (Acetylamino) -2-deoxy-β-D-galactopyranose

2- (Acetylamino) -2-deoxy-α-D-galactopyranose

In one aspect, the nucleic acid is a conjugated nucleic acid, wherein the nucleic acid has the following structure:

(In the formula, Z is any nucleic acid as defined herein).
It is conjugated to a tri-branched ligand having one of.

Preferably, the nucleic acid is a conjugated nucleic acid, wherein the nucleic acid has the following structure:

(In the formula, Z is any nucleic acid as defined herein).
It is conjugated to a trifurcated ligand having.

The ligand of any one of formula (II), formula (III) or formula (IV) or the trifurcated ligand disclosed herein is at the 3'end of the first (antisense) strand, and /. Or it can be attached to any of the 3'and / or 5'ends of the second (sense) strand. The nucleic acid may contain more than one of the formula (II), formula (III) or formula (IV) or any one of the trifurcated ligands disclosed herein. However, a single ligand of any one of formula (II), formula (III) or formula (IV) or the tri-branched ligand disclosed herein is preferred. This is because such a single ligand is sufficient for efficient targeting of nucleic acids to target cells. Preferably, in that case, at least the last two, preferably at least the last three, and more preferably at least the last four nucleotides at the end of the nucleic acid to which the ligand is bound are linked by a phosphodiester bond.

Preferably, the 5'end of the first (antisense) strand is of formula (II), formula (III) or formula (IV) or because the ligand at this position can potentially interfere with the biological activity of the nucleic acid. It does not bind to any one of the trifurcated ligands disclosed herein.

Nucleic acids having a single ligand of formula (II), formula (III) or formula (IV) at the 5'end of the chain or any one of the tri-branched ligands disclosed herein are the same at the 3'end. It is easier to synthesize than the same nucleic acid with a ligand and is therefore cheaper to synthesize. Thus, preferably, the single ligand of any one of formula (II), formula (III) or formula (IV) or any of the tri-branched ligands disclosed herein is the second strand of nucleic acid. Covalently (and conjugated) to the 5'end of.

In one aspect, the first strand of nucleic acid is the formula (V):

(In the formula, b is preferably 0 or 1)
Is a compound of
The second chain is Eq. (VI):

(During the ceremony,
c and d are independent, preferably 0 or 1,
Z ₁ and Z ₂ are the first and second strands of nucleic acid, respectively.
Y is independently O or S,
n is independently O, 1, 2 or 3,
L ₁ is a ligand-bound linker, where L ₁ is the same or different in equations (V) and (VI), and L ₁ is more than one in the same equation. In equations (V) and (VI), where many are present, the same or different, L ₁ is preferably equation (VII).
b + c + d is preferably 2 or 3)
It is a compound of.

Preferably, L ₁ in Eqs. (V) and Eq. (VI) is Eq. (VII) :.

(During the ceremony,
L is
-(CH ₂ ) _r -C (O)-(in formula, r = 2-12),
-(CH ₂ -CH ₂ -O) _s -CH ₂ -C (O)-(in the formula, s = 1-5),
-(CH ₂ ) _t -CO-NH- (CH ₂ ) _t -NH-C (O)-(in the equation, t is independent, 1-5),
-(CH ₂ ) _u -CO-NH- (CH ₂ ) _u -C (O)-(in the formula, u is 1 to 5 independently),
-(CH ₂ ) _v -NH-C (O)-(In the formula, v is 2 to 12)
, Or preferably selected from the group consisting of them,
Here, the terminus C (O) is present in X of Eq. (VII) if it is present, or in W ₁ of Eq. (VII) if X is not present, or if W ₁ is not present. Is coupled to V in equation (VII)
W ₁ , W ₃ and W ₅ individually do not exist or
-(CH ₂ ) _r- (in the formula, r = 1-7),
-(CH ₂ ) _s -O- (CH ₂ ) _s- (in the formula, s is independent, 0-5),
-(CH ₂ ) _t -S- (CH ₂ ) _t- (In the formula, t is 0 to 5 independently)
, Or preferably selected from the group consisting of them,
X is absent or is selected from the group consisting of NH, NCH ₃ or NC ₂ H ₅ or preferably consisting of them.
V is CH, N,

(In the formula, B is a modified or native nucleobase, if any)
, Or preferably selected from the group consisting of them)
Have.

In one aspect, the first strand is in equation (VIII).

(In the formula, b is preferably 0 or 1)
Is a compound of
The second chain is Eq. (IX):

(In the equation, c and d are independent, preferably 0 or 1, and are 0 or 1.
Z ₁ and Z ₂ are the first and second strands of nucleic acid, respectively.
Y is independently O or S,
R ₁ is H or methyl,
n is independent, preferably 0, 1, 2 or 3,
L is the same or different in equations (VIII) and (IX), and if more than one L is present in the same equation, then in equations (VIII) and (IX), Same or different,
-(CH ₂ ) _r -C (O)-(in formula, r = 2-12),
-(CH ₂ -CH ₂ -O) _s -CH ₂ -C (O)-(in the formula, s = 1-5),
-(CH ₂ ) _t -CO-NH- (CH ₂ ) _t -NH-C (O)-(in the equation, t is independent, 1-5),
-(CH ₂ ) _u -CO-NH- (CH ₂ ) _u -C (O)-(in the formula, u is 1 to 5 independently),
-(CH ₂ ) _v -NH-C (O)-(in the formula, v is 2-12)
, Or preferably selected from the group consisting of them,
Here, the terminal C (O), if present, is attached to an NH group (the NH group of the linker, not the NH group of the targeting ligand).
b + c + d is preferably 2 or 3)
It is a compound of.

In one aspect, the first strand of nucleic acid is the formula (X):

(In the formula, b is preferably 0 or 1)
The second chain is the compound of formula (XI):

(In the equation, c and d are independent, preferably 0 or 1, and are 0 or 1.
Z ₁ and Z ₂ are the first and second strands of nucleic acid, respectively.
Y is independently O or S,
n is independent, preferably 0, 1, 2 or 3,
L ₂ is the same or different in equations (X) and (XI), and is the same or different in the parts enclosed by b, c and d.

, Or preferably selected from the group consisting of them, or
n is 0 and L ₂ is

And the terminal OH group is absent so that the following moieties are formed:

(During the ceremony,
F is a saturated or unbranched (eg, unbranched) C _1-8 alkyl (eg, C _1-6 alkyl) chain, where one of the carbon atoms is optional. It is replaced with an oxygen atom, provided that the oxygen atom is separated from another heteroatom (eg, an O or N atom) by at least two carbon atoms.
L is the same or different in equations (X) and (XI),
-(CH ₂ ) _r -C (O)-(in formula, r = 2-12),
-(CH ₂ -CH ₂ -O) _s -CH ₂ -C (O)-(in the formula, s = 1-5),
-(CH ₂ ) _t -CO-NH- (CH ₂ ) _t -NH-C (O)-(in the equation, t is independent, 1-5),
-(CH ₂ ) _u -CO-NH- (CH ₂ ) _u -C (O)-(in the formula, u is 1 to 5 independently),
-(CH ₂ ) _v -NH-C (O)-(in the formula, v is 2-12)
, Or preferably selected from the group consisting of them, where the terminal C (O), if present, is attached to an NH group (the NH group of the linker, not the NH group of the targeting ligand). ,
b + c + d is preferably 2 or 3)
It is a compound of.

In one aspect, in any of the nucleic acids of formula (V) and formula (VI) or formula (VIII) and formula (IX) or formula (X) and formula (XI), b is 0 and c is 1. Yes, d is 1, b is 1, c is 0, d is 1, b is 1, b is 1, c is 1, d is 0, or b is 1 is 1, c is 1 and d is 1. Preferably, b is 0, c is 1, d is 1, b is 1, c is 0, d is 1, or b is 1, and c is 1. 1 and d is 1. Most preferably, b is 0, c is 1, and d is 1.

In one aspect, Y is O in any of the nucleic acids of formula (V) and formula (VI) or formula (VIII) and formula (IX) or formula (X) and formula (XI). In another aspect, Y is S. In a preferred embodiment, Y is independently selected from O or S at different positions in the equation.

In one aspect, R ₁ is H or methyl in any of the nucleic acids of formula (VIII) and formula (IX). In one aspect, R ₁ is H. In another aspect, R ₁ is methyl.

In one aspect, n is 0, 1, 2 or 3 in any of the nucleic acids of formula (V) and formula (VI) or formula (VIII) and formula (IX) or formula (X) and formula (XI). Is. Preferably n is 0.

Examples of the F portion in any of the nucleic acids of formula (X) and formula (XI) are (CH ₂ ) _1-6 , eg (CH ₂ ) _1-4 , eg CH ₂ , (CH ₂ ) ₄ , (CH 2) CH ₂ ) ₅ or (CH ₂ ) ₆ , or CH ₂ O (CH ₂ ) _2-3 , for example CH ₂ O (CH ₂ ) CH ₃ .

In one aspect, L ₂ in equation (X) and equation (XI) is

Is.

In one aspect, L ₂

Is.

In one aspect, L ₂

Is.

In one aspect, L ₂

Is.

In one aspect, n is 0 and L ₂ is

And the terminal OH group is absent so that the following moieties are formed:

(In the formula, Y is O or S).

In one aspect, L in the nucleic acids of formula (V) and formula (VI) or formula (VIII) and formula (IX) or formula (X) and formula (XI) is
-(CH ₂ ) _r -C (O)-(in formula, r = 2-12),
-(CH ₂ -CH ₂ -O) _s -CH ₂ -C (O)-(in the formula, s = 1-5),
-(CH ₂ ) _t -CO-NH- (CH ₂ ) _t -NH-C (O)-(in the equation, t is independent, 1-5),
-(CH ₂ ) _u -CO-NH- (CH ₂ ) _u -C (O)-(in the formula, u is 1 to 5 independently),
-(CH ₂ ) _v -NH-C (O)-(in the formula, v is 2-12)
, Or preferably selected from the group consisting of them, where the terminal C (O) is attached to an NH group.

Preferably, L is-(CH ₂ ) _r -C (O)-(in the equation, r = 2-12, more preferably r = 2-6, even more preferably r = 4 or 6, eg 4 ).

Preferably, L is

Is.

Within the part enclosed by b, c and d, L ₂ in the nucleic acids of formula (X) and formula (XI) is usually the same. In the parts enclosed by b, c and d, L ₂ can be the same or different. In some embodiments, L ₂ in the portion enclosed by c is the same as L ₂ in the portion enclosed by d. In some embodiments, L ₂ in the portion enclosed by c is not the same as L ₂ in the portion enclosed by d. In some embodiments, L ₂ in the moiety enclosed by b, c and d is the same, for example, if the linker moiety is a serinol-derived linker moiety.

The linker moiety derived from serinol can be based on any stereochemical serinol derived from the L-serine isomer, D-serine isomer, racemic serine or other combination of isomers. In a preferred embodiment of the invention, the serinol-GalNAc moiety (SerGN) is the stereochemistry of:

That is, it is based on the (S) -serinol-amidite or (S) -serine succinate solid support component derived from the L-serine isomer.

In a preferred embodiment, the first strand of nucleic acid is the compound of formula (VIII) and the second strand of nucleic acid is the compound of formula (IX), in the formula.
b is 0,
c and d are 1
n is 0,
Z ₁ and Z ₂ are the first and second strands of nucleic acid, respectively.
Y is S,
R ₁ is H,
L is-(CH ₂ ) ₄ -C (O)-where the terminus C (O) of L is attached to the N atom of the linker (ie, to the possible N atom of the targeting ligand). Do not combine).

In another preferred embodiment, the first strand of nucleic acid is the compound of formula (V) and the second strand of nucleic acid is the compound of formula (VI), in the formula.
b is 0,
c and d are 1
n is 0,
Z ₁ and Z ₂ are the first and second strands of nucleic acid, respectively.
Y is S,
L ₁ is Eq. (VII):
(During the ceremony,
W ₁ is -CH ₂ -O- (CH ₂ ) _3- ,
W ₃ is -CH _2- ,
W ₅ does not exist,
V is CH,
X is NH,
L is-(CH ₂ ) ₄ -C (O)-where the terminus C (O) of L is bonded to the N atom of X in equation (VII)).
Have.

In another preferred embodiment, the first strand of nucleic acid is the compound of formula (V) and the second strand of nucleic acid is the compound of formula (VI), in the formula.
b is 0,
c and d are 1
n is 0,
Z ₁ and Z ₂ are the first and second strands of nucleic acid, respectively.
Y is S,
L ₁ is Eq. (VII):
(During the ceremony,
W ₁ , W ₃ and W ₅ do not exist,
V is

And
X does not exist,
L is-(CH ₂ ) ₄ -C (O) -NH- (CH ₂ ) ₅ -C (O)-where the terminus C (O) of L is that of V in equation (VII). Bonded to N atom)
Have.

In one aspect, the nucleic acid has the following structure:

Conjugates to a tri-branched ligand with, where the nucleic acid is via the phosphate group of the ligand to the ligand, a) to the final nucleotide at the 5'end of the second strand, and b) to the 3 of the second strand. Conjugate to the final nucleotide at the'terminal end, or c) the final nucleotide at the 3'end of the first strand.

In one aspect of nucleic acid, the cell targeted by the nucleic acid carrying the ligand is a hepatocyte.

In any one of the above ligands in which GalNAc is present, GalNAc is substituted for any other targeting ligands such as those referred to herein, in particular mannose, galactose, glucose, glucosamine and fucose. You may.

A particularly preferred embodiment is a nucleic acid in which the first strand comprises or comprises SEQ ID NO: 356 and the second strand optionally comprises SEQ ID NO: 362 or comprises SEQ ID NO: 362. Is. This nucleic acid can be further conjugated to a ligand. Nucleic acids in which the first nucleic acid comprises or consists of SEQ ID NO: 356 and the second strand optionally comprises SEQ ID NO: 362 or consists of SEQ ID NO: 362 are more preferred. The siRNA consisting of SEQ ID NO: 356 and SEQ ID NO: 354 is most preferred. One aspect of the present invention is EV0203.

In one aspect, the nucleic acid is conjugated to a lipid-containing ligand, more preferably a cholesterol-containing ligand.

Compositions, Uses and Methods The invention also provides compositions comprising the nucleic acids of the invention. Nucleic acids and compositions can be used as pharmaceuticals or as diagnostic agents, alone or in combination with other agents. For example, one or more nucleic acids of the invention can be combined with delivery vehicles (eg, liposomes) and / or excipients, such as carriers, diluents. Other agents such as preservatives and stabilizers may also be added. Methods of delivery of nucleic acids are known in the art and are within the knowledge of one of ordinary skill in the art.

In one aspect, the composition comprises the nucleic acids and delivery vehicles disclosed herein and / or physiologically acceptable excipients and / or carriers and / or diluents and / or buffers and / or preservatives. Contains agents.

The nucleic acids of the invention or conjugated nucleic acids can also be administered separately or simultaneously, eg, in combination with other therapeutic compounds administered as a combined unit dose. The invention also comprises a composition comprising one or more nucleic acids according to the invention in physiologically / pharmaceutically acceptable excipients such as stabilizers, preservatives, diluents, buffers and the like. ..

In one aspect, the composition comprises the nucleic acids disclosed herein and additional therapeutic agents selected from the group comprising oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies and peptides. Preferably, the further therapeutic agent is an agent that targets, preferably inhibits, the expression or activity of another element of the complement component C3 or the immune system, or more specifically, a protein of the complement pathway. .. Preferably, the further therapeutic agent is: a) a peptide that inhibits the expression or activity of one of the components of the complement pathway, preferably one of C3 or C5 or one of those subunits, b) physiological. Antibodies that specifically bind to one of the components of the complement pathway, preferably either C3 or C5 or one of their subunits, under conditions, c) Eculizumab or one of their antigen-binding derivatives. It is one.

Eculizumab is a humanized monoclonal antibody that specifically binds to complement component C5 and is commercialized under the trade name SOLIRIS®. Eculizumab specifically binds complement component C5 with high affinity and inhibits the cleavage of C5 to C5a and C5b. Antibodies are described, for example, in European Patent No. 0 758 904 B1 and its family members.

Dosage levels for the pharmaceuticals and compositions of the invention can be determined by one of ordinary skill in the art through routine experimentation. In one aspect, the unit dose may contain from about 0.01 mg / kg to about 100 mg / kg (body weight) nucleic acid or conjugated nucleic acid. Alternatively, the dose is 10 mg / kg to 25 mg / kg (body weight), or 1 mg / kg to 10 mg / kg (body weight), or 0.05 mg / kg to 5 mg / kg (body weight), or 0.1 mg / kg to 5 mg / kg. It can be (body weight), or 0.1 mg / kg to 1 mg / kg (body weight), or 0.1 mg / kg to 0.5 mg / kg (body weight), or 0.5 mg / kg to 1 mg / kg (body weight). Alternatively, the dose is about 0.5 mg / kg to about 10 mg / kg (body weight), or about 0.6 mg / kg to about 8 mg / kg (body weight), or about 0.7 mg / kg to about 7 mg / kg (body weight), Or about 0.8 mg / kg to about 6 mg / kg (body weight), or about 0.9 mg / kg to about 5.5 mg / kg (body weight), or about 1 mg / kg to about 5 mg / kg (body weight), or about 1 mg / kg. It may be (body weight), or about 3 mg / kg (body weight), or about 5 mg / kg (body weight), where "about" is up to 30%, preferably up to 20% of the indicated value. Deviations of up to 10%, more preferably up to 5%, and most preferably up to 0%. Dosage levels can also be calculated by other parameters such as body surface area.

The pharmaceutical composition may also be a sterile, injectable aqueous suspension or solution, or may be present in lyophilized form.

The pharmaceutical compositions and pharmaceuticals of the present invention may be administered to a mammalian subject at a pharmaceutically effective dose. Mammals are selected from humans, non-human primates, monkeys or proto-monkeys, dogs, cats, horses, cows, pigs, goats, sheep, mice, rats, hamsters, hedgehogs and guinea pigs, or other related species. obtain. Based on this, "C3" as used herein means a nucleic acid or protein in any of the above species, when naturally or artificially expressed. , Preferably, this word means human nucleic acid or protein.

One aspect of the invention is the nucleic acid or composition disclosed herein for use as a pharmaceutical. The nucleic acid or composition is preferably for use in the prevention of a disease, disorder or syndrome, reduction of the risk of suffering from a disease, disorder or syndrome, or in the treatment of a disease, disorder or syndrome.

One aspect of the invention is a nucleic acid or composition as disclosed herein in the prevention of a disease, disorder or syndrome, the reduction of the risk of suffering from a disease, disorder or syndrome, or the treatment of a disease, disorder or syndrome. Is the use of.

One aspect of the invention prevents a disease, disorder or syndrome comprising administering a pharmaceutically effective dose of the nucleic acid or composition disclosed herein to an individual in need of treatment. A method of reducing the risk of suffering from a disease, disorder or syndrome, or treating a disease, disorder or syndrome, preferably where the nucleic acid or composition is subcutaneously and intravenously to the subject. , Or by oral, rectal, pulmonary or intraperitoneal administration. Preferably, the nucleic acid or composition is administered subcutaneously.

Diseases, disorders or syndromes to be prevented or treated with the nucleic acids or compositions disclosed herein are preferably complement-mediated diseases, disorders or syndromes, or diseases, disorders associated with the complement pathway. Or it is a syndrome.

Diseases, disorders or syndromes to be prevented or treated with the nucleic acids or compositions disclosed herein preferably include aberrant activation and / or overactivation (superactivation) of the complement pathway, as well as. / Or associated with overexpression or ectopic expression or localization or accumulation of complement component C3. An example of a disease involving the accumulation of C3 is C3 glomerulopathy (C3G). In this disease, C3 accumulates in the glomeruli of the kidney. Abnormalities or overactivation of the complement pathway to be prevented or treated can have a genetic cause or can be acquired. Preferably, the disease, disorder or syndrome to be prevented or treated is C3 glomerulopathy (C3G).

Diseases, disorders or syndromes to be prevented or treated with the nucleic acids or compositions disclosed herein are preferably a) C3 glomerulonephritis (C3G), paroxysmal nocturnal hemoglobinuria (PNH), unscripted. Lupus erythematosus syndrome (aHUS), lupus nephritis, IgA nephropathy (IgA N), lupus erythematosus (CAD), severe myasthenia (MG), primary membranous nephropathy, immune complex-mediated thread Glomerulonephritis (IC-mediated GN), post-infection glomerulonephritis (PIGN), systemic lupus erythematosus (SLE), ischemia-reperfusion injury, age-related luteal degeneration (AMD), rheumatoid arthritis (RA), antineupus erythematosus Autoantibody-related vasculitis (ANCA-AV), disbiosis periodontal disease, malaria anemia and septicemia, preferably selected from the group consisting of them, or b) C3 glomerulonephritis (C3G), paroxysmal Nocturnal hemoglobinuria (PNH), atypical lupus erythematosus syndrome (aHUS), lupus nephritis, IgA nephropathy (IgA N) and primary membranous nephropathy are included or preferably selected from the group consisting of them. Ruka or c) C3 glomerulonephritis (C3G), antineupus erythematosus autoantibody-related vasculitis (ANCA-AV), atypical lupus erythematosus syndrome (aHUS), lupus erythematosus (CAD), severe Whether it contains or is preferably selected from the group consisting of myasthenia (MG), IgA nephropathy (IgA N), paroxysmal nocturnal hemoglobinuria (PNH), or d) the disease, disorder or syndrome , C3 glomerulonephropathy (C3G). The subject to be treated with the nucleic acid or composition according to the invention is preferably the subject suffering from one of these diseases, disorders or syndromes.

The nucleic acids or compositions disclosed herein are weekly or twice weekly, weekly, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or eight. It may be for use in a regimen that includes weekly treatment or in a regimen with different dosing frequencies such as the combination of intervals described above. The nucleic acid or composition may be for use subcutaneously, intravenously, or using any other application route such as oral, rectal, lung or intraperitoneal. Preferably, the nucleic acid or composition is for subcutaneous use.

C3 expression is untreated in cells and / or subjects that have been treated with nucleic acids or compositions as disclosed herein or that receive nucleic acids or compositions as disclosed herein. 15% up to 100% compared to cells and / or subjects, but at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100%, or a range of intermediate values can be inhibited. The level of inhibition may allow treatment of diseases associated with C3 expression or overexpression or complement overactivation, or may help further study the function and physiological role of the C3 gene product.

One embodiment further comprises a disease, disorder or syndrome such as those listed above, or preferably elevated levels of C3 in the blood or kidney, or overactivation of the complement pathway, or inhibition of C3 expression. The use of nucleic acids or compositions as disclosed herein in the manufacture of a pharmaceutical for treating a further condition associated with a therapeutic approach.

Also, those listed above, including administration of a composition comprising a nucleic acid or composition as described herein to an individual in need of treatment (to ameliorate such pathology), etc. A method of treating or preventing a disease, disorder or syndrome of the above is included herein. Nucleic acid or composition twice a week, once a week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight to twelve weeks or it. It can be administered in a regimen that includes treatment for each of the above, or in a regimen that uses various dosing frequencies such as the combination of intervals described above. Nucleic acid or conjugated nucleic acid may be for use subcutaneously or intravenously, or by other application routes such as oral, rectal, or intraperitoneal.

The nucleic acids or compositions of the invention are produced using routine methods in the art, including chemical synthesis or expression of the nucleic acids either in vitro (eg, run-off transcription) or in vivo. Can be done. For example, solid-phase chemical synthesis is used, or nucleic acid-based expression, including viral derivatives or partially or fully synthetic expression systems, is used. In one aspect, the expression vector can be used to produce the nucleic acids of the invention in vitro, in an intermediate host organism or cell type, in an intermediate or final organism, or in a desired target cell. Methods of nucleic acid production (synthesis or enzymatic transcription) described herein are known to those of skill in the art.

The use of chemical modification patterns of nucleic acids confer nuclease stability in serum, for example making subcutaneous application pathways feasible.

The present invention is characterized by high specificity at the level of delivery directed at molecules and tissues, potentially conferring a better safety profile than currently available therapies.

Nucleic acids as described herein can be formulated with lipids in the form of liposomes. Such formulations may be described as lipoplexes in the art. Compositions using lipids / liposomes can be used to facilitate delivery of the nucleic acids of the invention to target cells. The lipid delivery system described herein can be used as an alternative to conjugated ligands. Modifications described herein may be present when using the nucleic acids of the invention with both lipid delivery systems or with ligand conjugate delivery systems.

Such a lipoplex
i) Cationic lipids, or pharmaceutically acceptable salts thereof,
ii) Steroids,
iii) Phosphatidylethanolamine phospholipids,
iv) Lipid compositions containing PEGylated lipids may be included.

The cationic lipid can be an amino cationic lipid.

Cationic lipids have the formula (XII):

Or may have a pharmaceutically acceptable salt thereof, in the formula,
X stands for O, S or NH
R ¹ and R ² independently represent C ₄ to C ₂₂ linear or branched alkyl chains or C ₄ to C ₂₂ linear or branched alkenyl chains with one or more double bonds, respectively. The alkyl or alkenyl chain optionally contains an intervening ester, amide or disulfide.
When X represents S or NH, R ³ and R ⁴ independently represent hydrogen, methyl, ethyl, mono or polyamine moieties, or R ³ and R ⁴ together form a heterocyclyl ring. ,
If X represents O, then R ³ and R ⁴ independently represent hydrogen, methyl, ethyl, mono or polyamine moieties, or R ³ and R ⁴ together form a heterocyclyl ring. Or R ³ represents hydrogen and R ⁴ represents C (NH) (NH ₂ ).

Cationic lipids have the formula (XIII):

Or may have a pharmaceutically acceptable salt thereof.

Cationic lipids have the formula (XIV):

Or may have a pharmaceutically acceptable salt thereof.

The content of the cationic lipid component can be from about 55 mol% to about 65 mol% of the total lipid content of the composition. In particular, the cationic lipid component is about 59 mol% of the total lipid content of the composition.

The composition may further comprise a steroid. The steroid may be cholesterol. The content of the steroid can be from about 26 mol% to about 35 mol% of the total lipid content of the lipid composition. More specifically, the content of the steroid can be about 30 mol% of the total lipid content of the lipid composition.

Phosphorylethanolamine Phosphorylethanolamines are 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), 1,2-dioreoil-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-di Stearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dimiristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dilinole oil-sn-glycero-3-phosphoethanolamine (DLoPE), 1-palmitoyle-2-ole Oil-sn-Glycero-3-phosphoethanolamine (POPE), 1,2-Dielcoyl-sn-Glycero-3-phosphoethanolamine (DEPE), 1,2-Dissuare Oil-sn-Glycero-3-phosphoethanol It can be selected from the group consisting of amine (DSQPE) and 1-stearoyl-2-linole oil-sn-glycero-3-phosphoethanolamine (SLPE). The content of phospholipids can be about 10 mol% of the total content of lipids in the composition.

The pegylated lipid can be selected from the group consisting of 1,2-dimyristyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG) and C16-ceramide-PEG. The content of the pegulated lipid can be from about 1 mol% to 5 mol% of the total content of the lipid in the composition.

The content of the cationic lipid component in the composition can be from about 55 mol% to about 65 mol% of the total lipid content of the lipid composition, preferably about 59 mol% of the total lipid content of the lipid composition.

The compositions are 55:34:10: 1, 56:33:10: 1, 57:32:10: 1, 58:31:10: 1, 59:30:10: 1, 60:29:10: Choose from 1, 61: 28: 10: 1, 62: 27: 10: 1, 63: 26: 10: 1, 64: 25: 10: 1, and 65: 24: 10: 1 i): ii ): iii): iv) may have a molar ratio of components.

The composition is structural

Cationic lipids with

Construction

Steroids with

Construction

Phosphatidylethanolamine phospholipids with

And structure

May include pegylated lipids having.

The neutral liposome composition can be formed, for example, from dimyristoylation phosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC). Anionic liposome compositions can be formed from dimyristylphosphatidylglycerol, whereas anionic membrane fusion liposomes can be formed primarily from dioleoylphosphatidylethanolamine (DOPE). Another type of liposome composition can be formed from phosphatidylcholine (PC), such as soybean PC, egg PC, and the like. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

Spontaneously interact with nucleic acids using N- [1- (2,3-diorail oil) propyl] -N, N, N-trimethylammonium chloride (DOTMA), a positively charged synthetic cationic lipid. It is possible to form working small liposomes and form lipid-nucleic acid complexes capable of fusing with negatively charged lipids in the cell membrane of tissue culture cells. DOTMA analogs can also be used to form liposomes.

Lipid derivatives and analogs described herein can also be used to form liposomes.

Liposomes containing nucleic acids can be prepared by a variety of methods. In one example, the lipid component of the liposome is dissolved in the detergent, resulting in the formation of micelles with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or a lipid conjugate. The cleaning agent can have a high critical micelle concentration and may be nonionic. Exemplary cleaning agents include cholic acid salt, CHAPS, octyl glucoside, deoxycholic acid salt, and lauroyl sarcosine. The nucleic acid preparation is subsequently added to the micelle containing the lipid component. Cationic groups on the lipid interact with the nucleic acid and condense around the nucleic acid to form liposomes. After condensation, the detergent is removed, for example by dialysis, to give a liposome preparation of nucleic acid.

If desired, carrier compounds useful for condensation can be added during the condensation reaction, for example by controlled addition. For example, the carrier compound can be a polymer other than nucleic acid (eg, spermine or spermidine). The pH can also be adjusted to support condensation.

Nucleic acid formulations may include detergents. In one embodiment, the nucleic acid is formulated as an emulsion containing a surfactant.

The non-ionized surfactant is a nonionic surfactant. Examples include nonionic esters such as ethylene glycol esters, polypropylene glycol esters, glyceryl esters, nonionic alkanolamides, and ethers such as fatty alcohol ethoxylates, propoxylated alcohols and ethoxylated / propoxylated block polymers. Be done.

Surfactants that retain a negative charge when dissolved or dispersed in water are anionic surfactants. Examples include carboxylates such as soaps, acyllactylates, acylamides of amino acids, esters of sulfuric acid such as alkylsulfates and ethoxylated alkylsulfates, sulphonates such as alkylbenzenes sulfonates, acylisetionates, acyltaurates and sulfosuccinates. , And phosphate.

Surfactants that retain a positive charge when dissolved or dispersed in water are cationic surfactants. Examples include quaternary ammonium salts and ethoxylated amines.

A surfactant having the ability to retain either a positive or negative charge is an amphoteric surfactant. Examples include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

A "micelle" is a specific arrangement of amphipathic molecules in a spherical structure such that all hydrophobic parts of the molecule are oriented inward and the hydrophilic parts are in contact with the surrounding aqueous phase. As defined herein as a type of molecular assembly. If the environment is hydrophobic, the reverse arrangement exists. Micelle can be formed by mixing an aqueous solution of nucleic acid, alkali metal alkyl sulfate, and at least one micelle-forming compound.

Exemplary micelle-forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monooleine, monooleate. , Monolaurate, Ruridisa Oil, Matsuyoigusa Oil, Mentor, Trihydroxyoxocholanylglycine and its pharmaceutically acceptable salts, glycerol, polyglycerol, lysine, polylysine, triolein, polyoxyethylene ethers and their analogs. , Polydocanolalkyl ethers and their analogs, chenodeoxycholic acid salts, deoxycholic acid salts, and mixtures thereof.

Phenol and / or m-cresol can be added to the mixed micelle composition to act as stabilizers and preservatives. An isotonic agent such as glycerin may be added.

The nucleic acid preparation may be incorporated into particles such as fine particles. Fine particles can be produced by spray drying, freeze drying, evaporation, fluidized bed drying, vacuum drying, or a combination of these methods.

Definitions As used herein, with respect to gene expression, the terms "inhibit,""down-regulate," or "reduce" are the expression of a gene, or one or more proteins or protein subunits. The level of the RNA molecule encoding or equivalent RNA molecule (eg, mRNA), or the activity of one or more proteins or protein subunits is observed in the absence of the nucleic acids of the invention or conjugated nucleic acids. When compared to those obtained using siRNA molecules that are less than or have no known homology to human transcripts (referred to herein as non-silenced controls). It means that it will be reduced. Such controls can be conjugated and modified in a manner similar to the molecules of the invention and delivered to target cells by the same pathway. Expression after treatment with the nucleic acids of the invention is 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 15%, or above the median. Or less than what is observed in the absence of nucleic acid or conjugated nucleic acid. Expression can be measured in cells to which the nucleic acid is applied. Alternatively, levels can be measured in various groups of cells, or in tissues or organs, or in body fluids such as blood or plasma, especially when nucleic acids are administered to the subject. The level of inhibition is preferably measured in vitro under the conditions selected so that the conditions show the maximum effect of the nucleic acid on the target mRNA level in the cells treated with the nucleic acid. The level of inhibition can be measured, for example, between 0.038 nM and 10 μM, preferably 24 hours or 48 hours after treatment with nucleic acid at concentrations of 1 nM, 10 nM or 100 nM. These conditions may differ for different nucleic acid sequences, such as unmodified or modified nucleic acids, or nucleic acids conjugated or unconjugated to ligands, or different types of nucleic acids. Examples of suitable conditions for determining the level of inhibition are described in the Examples.

Nucleic acid means a nucleic acid containing two strands containing a nucleotide capable of interfering with gene expression. Inhibition may be complete or partial, resulting in downregulation of gene expression in a targeted manner. The nucleic acid comprises a first strand of two separate polynucleotide strands, which can also be a guide strand, and a second strand, which can also be a passenger strand. The first and second strands may be part of the same self-complementary polynucleotide molecule that "folds back" to form a double chain molecule. The nucleic acid may be a siRNA molecule.

Nucleic acids include ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analog non-nucleotides capable of mimicking nucleotides so that they can "pair" with the corresponding base on the target sequence or complementary strand. Can include. Nucleic acid is formed by all or part of the first strand (also known as a guide strand in the art) and all or part of the second strand (also known as a passenger strand in the art). It may further contain a double-stranded nucleic acid moiety or a double-stranded region. The double-stranded region begins with the first base pair formed between the first and second strands and the last formed between the first and second strands. Defined as ending in base pair (including both ends).

Double-stranded regions are base paired with each other by either Watson-Crick base pairing or any other mode that allows double-strands between complementary or substantially complementary oligonucleotide chains. Means a region in two complementary or substantially complementary oligonucleotides that form. For example, an oligonucleotide chain with 21 nucleotide units can base pair another oligonucleotide with 21 nucleotide units, on which the "double-stranded region" is from 19 base pairs. As such, only 19 nucleotides on each strand are complementary or substantially complementary. The remaining base pairs can exist as 5'overhangs and 3'overhangs, or as single chain regions. Moreover, within the double-stranded region, 100% complementarity is not required and substantial complementarity is possible within the double-stranded region. Substantial complementarity refers to complementarity between chains such that the chains can be annealed under biological conditions. Techniques for empirically determining whether two strands can be annealed under biological conditions are well known in the art. Alternatively, the two chains can be synthesized and added together under biological conditions to determine if they are annealing to each other. The first and second strand portions forming at least one double-stranded region may be completely complementary or at least partially complementary to each other. Depending on the length of the nucleic acid, a perfect match for base complementarity between the first strand and the second strand is not always required. However, the first and second chains must be capable of hybridizing under physiological conditions.

As used herein, the term "unpaired nucleotide analog" refers to 6-desaminoadenosine (nebralin), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3. -Me ribo U, N3-Me ribo T, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, and N3-Me dC, but these Means a nucleotide analog containing a non-base pairing moiety, not limited to. In some embodiments, the non-base pairing nucleotide analog is a ribonucleotide. In other embodiments, the non-base pairing nucleotide analog is a deoxyribonucleotide.

As used herein, the term "terminal functional group" includes, but includes, a halogen group, an alcohol group, an amine group, a carboxy group, an ester group, an amide group, an aldehyde group, a ketone group, and an ether group. Not limited to.

"Overhang", as used herein, has its general and customary meaning in the art, i.e., a single nucleic acid that extends beyond the terminal nucleotide of the complementary strand in a double-stranded nucleic acid. It is a chain part. The term "blunt-ended" includes double-stranded nucleic acids in which both strands terminate at the same position regardless of whether the terminal nucleotide is base paired. The terminal nucleotides of the first and second strands at the blunt ends may be base paired. The terminal nucleotides of the first and second strands at the blunt ends do not have to be base paired. The two nucleotides at the ends of the first and second strands at the blunt ends may be base paired. The two nucleotides at the ends of the first and second strands at the blunt ends do not have to be base paired.

The term "linker moiety derived from serinol" means that the linker moiety has the following structure:

Means to include.

The O atom of the above structure is usually linked to the RNA strand, and the N atom is usually linked to the targeting ligand.

Here, the invention is described with reference to the following non-limiting drawings and examples.

It is a figure which shows the concentration-response-curve of selected siRNA. It is a figure which shows the concentration-response-curve of selected siRNA. It is a figure which shows the concentration-response-curve of selected siRNA. It is a figure which shows the concentration-response-curve of the selected siRNA GalNAc conjugate in the human primary cultured hepatocyte. It is a figure which shows the concentration-response-curve of the selected siRNA GalNAc conjugate in the human primary cultured hepatocyte. It is a figure which shows the concentration-response-curve of the selected siRNA GalNAc conjugate in the mouse primary cultured hepatocyte. It is a figure which shows the concentration-response-curve of the selected siRNA GalNAc conjugate in the mouse primary cultured hepatocyte. 4A, 4B and 4C are diagrams showing concentration-dependent C3 mRNA inhibition of selected siRNA GalNAc conjugates in primary cultured mouse, human and cynomolgus monkey hepatocytes, respectively. It is a figure which shows the possible synthetic pathway to DMT-serinol (GalNAc) -CEP and CPG. FIG. 5 shows in vivo C3 mRNA levels in hepatocytes in response to mouse sc treatment with selected siRNA GalNAc conjugates. FIG. 5 shows serum C3 protein levels in response to mouse sc treatment with selected siRNA GalNAc conjugates. 7A, 7B and 7C show primary cultured mice (A), cynomolgus monkeys (B) and humans after incubation with selected siRNA GalNAc conjugates (1nM, 10nM and 100nM) normalized to ACTIN mRNA. (C) It is a figure which shows the relative C3 mRNA expression in a hepatocyte. 8A and 8B are relative in% of mouse liver 14 days (A) or 42 days (B) after a single dose of GalNAc-conjugated siRNAs EV0201, EV0203, EV0204, EV0205 and EV0207. It is a figure which shows the expression of C3 mRNA. The data are shown in the bar graph as mean ± SD (n = 4 per group). FIGS. 9A-9E show (BL) before 5 mg / kg or 10 mg / kg siRNA dosing, and on the 4th, 10th, and 14th days of the study after 5 mg / kg or 10 mg / kg siRNA dosing. It is a figure which shows the relative C3 protein serum level in% from the mouse serum sample taken on day, day 21, day 28, day 35, and day 42. Data points represent individual animal serum C3 levels as determined using standard C3 ELISA. The data were normalized to the baseline mean for each group, followed by a PBS control that matched the time set to 100%. The plotted lines connect the mean values of the individual groups at each time point. A) Normalized C3 serum levels after administration of 5 mg / kg and 10 mg / kg EV0201, B) Normalized C3 serum levels after administration of 5 mg / kg and 10 mg / kg EV0203, C) 5 mg / kg Normalized C3 serum levels after administration of kg EV0204, D) normalized C3 serum levels after administration of 5 mg / kg and 10 mg / kg EV0205, E) of EV0207 at 5 mg / kg and 10 mg / kg Normalized C3 serum levels after dosing. Figure 9F shows dosing with (BL), 0.3 mg / kg, 1 mg / kg, 3 mg / kg or 5 mg / kg siRNA prior to dosing with 0.3 mg / kg, 1 mg / kg, 3 mg / kg or 5 mg / kg siRNA. It is a figure which shows the relative C3 protein serum level (in%) from the mouse serum sample taken on the 7th day, the 14th day, the 21st day and the 28th day of a later study.

(Example 1)
In vitro studies in HepG2 cells demonstrating the C3 mRNA knockdown efficacy of the siRNA tested after transfection with 10 nM siRNA.
The C3 knockdown efficacy of siRNA EV0001-EV0100 was determined after transfection of 10nM siRNA in HepG2 cells. The results are shown in Table 2 below. Residual C3 mRNA levels after knockdown ranged from 6% to 83%. The most potent siRNAs were EV0001, EV0007, EV0008, EV0009, EV0012, EV0013, EV0018, EV0020, EV0030, EV0033, and EV0004.

For transfection of HepG2 with siRNA, cells were seeded on a collagen-coated 96-well tissue culture plate (# 655150, GBO, Germany) at a density of 15,000 cells / well. Transfection of siRNA was performed using Lipofectamine RNAiMax (Invitrogen / Life Technologies, Karlsruhe, Germany) immediately after seeding according to the manufacturer's instructions. Screening was performed in quadruple iterations with C3 siRNA at 10 nM using siRNA targeting Aha1, firefly luciferase and Factor VII as non-specific controls and pseudotransfections. After 24 hours of incubation with siRNA, medium was removed and cells were lysed in 150 μl of medium-dissolved mixture (1x volume of lytic mixture, 2x volume of cell culture medium) and then incubated at 53 ° C. for 30 minutes. The bDNA assay was performed according to the manufacturer's instructions. Luminescence was read in the dark at RT 30 minutes after incubation using a 1420 Luminescence Counter (WALLAC VICTOR Light, Perkin Elmer, Rodgau-Jügesheim, Germany). For each well, C3 mRNA levels were normalized to each GAPDH mRNA level. The activity of a given C3 siRNA is the percentage of residual C3 mRNA concentration (normalized for GAPDH mRNA) in treated cells relative to the C3 mRNA concentration averaged across control wells (normalized for GAPDH mRNA). Expressed as.

Table 2: C3 siRNA screening results-single-stranded identities forming each siRNA duplex, as well as their sequences and modifications, can be found in the table at the end of the description.

(Example 2)
In vitro studies in HepG2 cells demonstrating the C3 mRNA knockdown efficacy of siRNA tested after transfection of 0.01 pM-100 nM siRNA (concentration-response-curve experiment).
The C3 knockdown efficacy of siRNA EV0001, EV0008, EV0013, EV0030, EV0033, EV0039, EV0043, EV0053, EV0054, EV0059, EV0060, EV0061, EV0066, EV0072, EV0075, EV0081, EV0091 and EV0098 is 0.01pM in HepG2 cells. Determined after transfection of ~ 100 nM siRNA. The single-stranded identities that form each siRNA duplex and their sequences can be found in the table at the end of the description. The results are presented in FIGS. 1A, 1B and 1C. All siRNAs showed dose-dependent knockdown of C3 mRNA after transfection. The most potent siRNAs were EV0008, EV0033 and EV0081, with residual C3 expression of 100 nM siRNA of 4.3%, 5.3% and 5.3%, respectively.

For transfection of HepG2 with siRNA, cells were seeded on a collagen-coated 96-well tissue culture plate (# 655150, GBO, Germany) at a density of 15,000 cells / well. Transfection of siRNA was performed using Lipofectamine RNAiMa (Invitrogen / Life Technologies, Karlsruhe, Germany) immediately after seeding according to the manufacturer's instructions. Concentration-response experiments were performed using C3 siRNA at 10 concentrations transfected in quadruple iteration, starting at 100 nM and up to 0.01 pM in the 6-fold dilution step. Pseudo-transfected cells served as a control in CRC experiments. After 24 hours of incubation with siRNA, medium was removed and cells were lysed in 150 μl of medium-dissolved mixture (1x volume of lytic mixture, 2x volume of cell culture medium) and then incubated at 53 ° C. for 30 minutes. The bDNA assay was performed according to the manufacturer's instructions. Luminescence was read in the dark at RT 30 minutes after incubation using a 1420 Luminescence Counter (WALLAC VICTOR Light, Perkin Elmer, Rodgau-Jügesheim, Germany). For each well, C3 mRNA levels were normalized to each GAPDH mRNA level. The activity of a given C3 siRNA is as a percentage of the remaining C3 mRNA (normalized for GAPDH mRNA) in treated cells to the C3 mRNA concentration averaged across control wells (normalized for GAPDH mRNA). expressed. Concentration-response curves were fitted using GraphPad Prism version 7.05 using a 4-parameter logistic (4PL) model without further constraints.

(Example 3)
In vitro studies in primary cultured human hepatocytes demonstrating the C3 mRNA knockdown efficacy of siRNA-GalNAc conjugates (0.038 nM-10 μM siRNA conjugates) tested in a concentration-response-curve format.
Expression of C3 mRNA after incubation with GalNAc siRNA conjugates EV0101, EV0102, EV0103, EV0104, EV0105, EV0106, EV0107, EV0108, EV0109, EV0110, EV0111 and EV0312 was analyzed in a concentration-response format. The single-stranded identities that form each siRNA duplex and their sequences can be found in the table at the end of the description. The results are shown in FIGS. 2A and 2B. The mRNA levels of the housekeeping gene GAPDH served as a control for all experiments. All siRNA GalNAc conjugates were able to reduce C3 mRNA levels in a concentration-dependent manner, with maximal inhibition at 10 μM of 32% to 70%, respectively. The most potent siRNAs were EV102 with a 61% reduction in C3 mRNA levels at 10 μM, EV109 with a 67% reduction, EV0111 with a 69% reduction, and EV0302 with a 70% reduction.

Human cryopreserved primary cultured hepatocytes were purchased from Primacyt (Schwerin, Germany, cat # GuCPI, Lot # BHum16061-P). Immediately prior to treatment, cells were thawed, transferred to a tube with thawing medium (Primacyt, cat # HTM), centrifuged and washed with wash medium (Primacyt, cat # HWM). Cells are placed in a plate culture medium (Primacyt, cat # HPM-cryo) on a collagen-coated 96-well plate (Greiner-Bio-One, # 655150) with a density of 90,000 cells per well. Sown. Immediately after seeding, the cells adhered as a monolayer in plate culture medium, so the cells were treated with siRNA. Each siRNA was applied to cells in terms of concentration-response-curve at concentrations starting at 10 μM and serially diluted to 38 pM in 4-fold dilution steps. Each concentration was applied as a quadruple repeat. After 5 hours, the medium was replaced with maintenance medium (Primacyt, cat # HHMM). Medium was changed every 24 hours and cells were collected 48 hours after seeding for analysis by the Quantigene bDNA assay. C3 mRNA concentration was normalized to GAPDH mRNA. Concentration-response-curves were fitted using GraphPad Prism version 7.05 using a 4-parameter logistic (4PL) model without further constraints.

(Example 4)
In vitro studies of primary cultured mouse hepatocytes demonstrating the C3 mRNA knockdown efficacy of siRNA-GalNAc conjugates (0.038 nM-10 μM siRNA conjugates) tested in a concentration-response-curve format.
Expression of C3 mRNA after incubation with GalNAc siRNA conjugates EV0104, EV0105, EV0107, EV0108, EV0109, EV0110, EV0111 and EV0312 in a concentration-response format was analyzed. The single-stranded identities that form each siRNA duplex and their sequences can be found in the table at the end of the description. The mRNA levels of the housekeeping gene GAPDH served as a control. The siRNA GalNAc conjugate was able to reduce C3 mRNA levels in a concentration-dependent manner, with maximal inhibition at 10 μM ranging from 56% to 72%. The most potent siRNAs were EV0104 with 68% reduction in C3 mRNA at 10 μM, EV0109 with 67% reduction, and EV0111 with 72% reduction, respectively.

Cryopreserved mouse hepatocytes are purchased from Thermo Fisher (# MSCP10, Lot # MC817) and are in plate culture medium (William E medium, no phenol red-total 500 ml, Thermo Fisher Sci, Cat. No. A12176-01). Sowed in Thermo Fisher Sci, Cat. No. CM3000 replenishment pack) added to. On the day of sowing, the cells were thawed and sown on a collagen-coated 96-well plate (Greiner-Bio-One, # 655150) at a density of 60,000 cells per well. Immediately after seeding, the cells adhered as a monolayer in plate culture medium, so the cells were treated with siRNA. Each siRNA was applied to cells as a concentration-response-curve at concentrations starting at 10 μM and serially diluted to 0.038 nM in 4-fold dilution steps. Each concentration was applied as a quadruple repeat. After 5 hours, the medium was added to maintenance medium (Thermo Fisher Sci, William E medium, no phenol red-total 500 ml, Thermo Fisher Sci, Cat. No. A12176-01, Thermo Fisher Sci Cat. No. Replaced with CM4000 replenishment pack). Medium was changed every 24 hours and cells were collected 48 hours after seeding for analysis by the Quantigene bDNA assay.

The results are shown in FIGS. 3A and 3B. The results represent the percentage of residual C3 mRNA expression in primary cultured mouse hepatocytes after incubation with siRNA GalNAc conjugate (0.038 nM-10 μM) normalized to GAPDH mRNA. Concentration-response curves were fitted using GraphPad Prism version 7.05 using a 4-parameter logistic (4PL) model without further constraints.

(Example 5)
In vitro studies in primary cultured mouse, human and cynomolgus monkey hepatocytes showing c3 mRNA knockdown of 1nM, 10nM and 100nM tested siRNA-GalNAc conjugates.
Expression of c3 mRNA after incubation with 1 nM, 10 nM and 100 nM GalNAc siRNA conjugates EV0312 and EV0313 was analyzed. The single-stranded identities that form each siRNA duplex and their sequences can be found in the table at the end of the description. The mRNA levels of the housekeeping gene actin served as a control.

40,000 (human), 30,000 (mouse) or 45,000 (cynomolgus monkey) cells were seeded on collagen-coated 96-well plates. The indicated concentration of siRNA was added immediately after seeding. Twenty-four hours after treatment, cells were lysed using the InviTrap RNA Cell HTS96 Kit / C (Stratec). QPCR was performed using mRNA-specific primers and probes for C3 and actin.

The results are shown in FIGS. 4A, 4B and 4C.

(Example 6)
Component Synthesis The synthetic pathways for DMT-serinol (GalNAc) -CEP and CPG as described below are outlined in Figure 5. The starting material DMT-serinol (H) (1) was made from commercially available L-serine according to the method published in the literature (Hoevelmann et al., Chem. Sci., 2016, 7, 128-135). GalNAc (Ac ₃ ) -C ₄ H ₈ -COOH (2) is a method published in the literature (Nair et al., J. Am. Chem. Soc., 2014, 136 (49), pp. 16958-1696). According to, it was prepared starting from a commercially available peracetylated galactose amine. Phosphytylation Reagent 2-Cyanoethyl-N, N-diisopropylchlorophosphoruamideite (4) is commercially available. (vp)-mU-phos synthesis is described in Prakash, Nucleic Acids Res., 2015, 43 (6), pp. 2993-3011 and Haraszti, Nucleic Acids Res., 2017, 45 (13), pp. 7581-7592. It was carried out as described in. The synthesis of the phosphoramidite derivatives of ST43 (ST43-phos) and ST23 (ST23-phos) can be carried out as described in WO 2017/174657.

DMT-Serinol (GalNAc) (3)
HBTU (9.16 g, 24.14 mmol) was added to a stirred solution of GalNAc (Ac ₃ ) -C ₄ H ₈ -COOH (2) (11.4 g, 25.4 mmol) and DIPEA (8.85 ml, 50.8 mmol). After an activity time of 2 minutes, a solution of DMT-serinol (H) (1) (10 g, 25.4 mmol) in acetonitrile (anhydrous) (200 ml) was added to the stirred mixture. After 1 hour, LCMS showed good conversion. The reaction mixture was concentrated in vacuo. The residue was dissolved in EtOAc and subsequently washed with water (twice) and saline. The organic layer was dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The residue was further purified by column chromatography (3% MeOH in CH ₂ Cl ₂ + 1% Et ₃ N, 700 g silica). The product-containing fractions are pooled, concentrated, stripped with CH ₂ Cl ₂ (twice), and DMT-serinol (GalNAc) (3) 10.6 g (51) as an off-white foam. %) Was obtained.

DMT-Serinol (GalNAc) -CEP (5)
2-cyanoethyl-N, N-diisopropylchlorophosphoramidite (4) (5.71 ml, 25.6 mmol) in DMT-serinol (GalNAc) (3) (15.0 g, 17.0 mmol) in dichloromethane (dried) (150 ml) , DIPEA (14.9 ml, 85 mmol) and 4 Å of molecular sieves were added slowly at 0 ° C. under an argon atmosphere. The reaction mixture was stirred at 0 ° C. for 1 hour. TLC showed the complete conversion. The reaction mixture was filtered and concentrated in vacuo to give a thick oil. The residue was dissolved in dichloromethane and further purified by flash chromatography (0% -50% acetone in 1% toluene ET3N, 220 g silica). Product-containing fractions were pooled and concentrated in vacuo. The obtained oil was removed with MeCN (twice) to give 13.5 g (77%) of colorless DMT-serinol (GalNAc) -CEP (5) foam.

DMT-Serinol (GalNAc) -Succinate (6)
DMAP (1.11 g, 9.11 mmol), DMT-serinol (GalNAc) (3) (7.5 g, 9.11 mmol) and succinic anhydride (4.56 g, 45.6 mmol) in a mixture of dichloromethane (50 ml) and pyridine (50 ml). Was added to the stirred solution of Succinic anhydride under an atmosphere of argon. After 16 hours of stirring, the reaction mixture was concentrated in vacuo and the residue was dissolved in EtOAc and washed with 5% citric acid (aq). The aqueous layer was extracted with EtOAc. Subsequently, the combined organic layer was then washed with saturated NaHCO ₃ (aq.) And saline, dried over Na ₂ SO ₄ , filtered and concentrated in vacuo. Further purification was achieved by flash chromatography (0% -5% MeOH in CH ₂ Cl ₂ + 1% ET ₃ N, 120 g silica). Product-containing fractions were pooled and concentrated in vacuo. The residue was dropped with MeCN (3 times) to give DMT-serinol (GalNAc) -succinate (6) 5.9 g (70%).

DMT-Serinol (GalNAc) -Succinyl-lcaa-CPG (7)
DMT-serinol (GalNAc) -succinate (6) (1 eq) and HBTU (1.1 eq) were dissolved in CH ₃ CN (10 ml). Diisopropylethylamine (2 eq) was added to the solution, the mixture was rotated for 2 minutes and then natural amino-lcaa-CPG (500 A, 88 μmol / g, 1 eq) was added. The suspension was gently shaken on a wrist action shaker at room temperature for 16 hours, then filtered and washed with acetonitrile. The solid support was dried under reduced pressure for 2 hours. Unreacted amines on the support were capped with Ac ₂ O / 2,6-lutidine / NMI by stirring at room temperature (15 minutes x 2 times). Cleaning of the support was repeated as described above. The solid was dried under vacuum to give DMT-serinol (GalNAc) -succinyl-lcaa-CPG (7) (load: 34 μmol / g, determined by detritylization assay).

(Example 7)
Oligonucleotide Synthesis Examples of the compounds were synthesized according to the methods described below and methods known to those of skill in the art. The construction of oligonucleotide chains and linker components was carried out by solid phase synthesis applying the phosphoramidite methodology.

Downstream cleavage, deprotection and purification followed standard procedures known in the art.

Oligonucleotide synthesis was performed on AKTA oligopilot 10 using commercially available 2'O-methyl RNA and 2'fluoro-2' deoxy RNA base-added CPG solid supports, and phosphoramidite (all standard protection). , ChemGenes, LinkTech) was used. The synthesis of DMT- (S) -serinol (GalNAc) -succinyl-lcaa-CPG (7) and DMT- (S) -serinol (GalNAc) -CEP (5) is described in Example 6.

Auxiliary reagents were purchased from EMP Biotech. Synthesis was carried out using a 0.1 M solution of phosphoramidite in dry acetonitrile (<20 ppm H ₂ O) and benzylthiotetrazole (BTT) was used as an activator (0.3 M in acetonitrile). The coupling time was 10 minutes. A Cap / OX / Cap or Cap / Thio / Cap cycle was applied (Cap: Ac ₂ O / NMI / lutidine / acetonitrile, oxidant: 0.05 MI ₂ in pyridine / H ₂ O). Phosphorothioates were introduced using a commercially available thiolation reagent 50 mM EDITH (Link technologies) in acetonitrile. DMT cleavage was achieved by treatment with 3% dichloroacetic acid in toluene. Diethylamine (DEA) washes were performed at the completion of the programmed synthesis cycle. All oligonucleotides were synthesized in DMT-off mode.

The binding of the serinol (GalNAc) moiety is either base loaded (S) -DMT-serinol (GalNAc) -succinyl-lcaa-CPG (7) or (S) -DMT-serinol (GalNAc) -CEP (5). Achieved by the use of. The trifurcated GalNAc cluster (ST23 / ST43) was introduced by continuous coupling of a bifurcated trebrer amidite derivative (C6XLT-phos) followed by GalNAc amidite (ST23-phos). Binding of the (vp) -mU moiety was achieved by the use of (vp) -mU-phos in the final synthetic cycle. (vp) -mU-phos do not provide a suitable hydroxy group for further synthetic extension and therefore do not carry a DMT group. Therefore, the coupling of (vp) -mU-phos results in a synthetic termination.

For the removal of the methyl ester masking vinyl phosphonate, the CPG carrying the fully constructed oligonucleotide was dried under reduced pressure to a 20 ml PP syringe reactor (Carl) for solid phase peptide synthesis with disk frit. Moved to Roth). The CPG was then contacted with a solution of TMSBr 250 μl and pyridine 177 μL in CH ₂ Cl ₂ at room temperature (0.5 ml / μmol solid support binding oligonucleotide) and the reactor was sealed with a Luer cap. The reaction vessel was stirred slightly over a period of 15 minutes x 2 times, the excess reagent was discarded, and the residual CPG was washed twice with 10 ml of acetonitrile. Further downstream processing did not change from any other exemplary compound.

Single chains were cleaved from CPG by treatment with 40% methylamine water (90 minutes, RT). The resulting crude oligonucleotide was purified by ion exchange chromatography (Resource Q, 6 ml, GE Healthcare) using a sodium chloride gradient on the AKTA Pure HPLC system. Product-containing fractions were pooled, desalted on a size exclusion column (Zetadex, EMP Biotech) and lyophilized for further use.

All final single chain products were analyzed by AEX-HPLC to prove their purity. The identity of each single chain product was demonstrated by LC-MS analysis.

(Example 8)
Double chain formation Individual single chains were dissolved in H ₂ O at a concentration of 60 OD / ml. Both individual oligonucleotide solutions were added together in the reaction vessel. Titration was performed for easier reaction monitoring. The first strand was added 25% more than the second strand, as determined by UV absorption at 260 nm. The reaction mixture was heated to 80 ° C. for 5 minutes and then gradually cooled to RT. Double chain formation was monitored by ion pairing reverse phase HPLC. From the UV area of the residual single chain, the required amount of the second chain was calculated and added to the reaction mixture. The reaction was heated again to 80 ° C. and gradually cooled to RT. This procedure was repeated until less than 10% of the residual single chain was detected.

(Example 9)
An in vivo study showing knockdown of C3 mRNA and serum protein in mouse liver tissue after a single subcutaneous dose of 1 mg / kg or 5 mg / kg GalNAc conjugated siRNA.
Eight-week-old female C57BL / 6N mice were obtained from CHARLES RIVER, Sulzfeld, Germany. Animal studies were performed in accordance with the July 2013 version of the German Protection of Animals Act ethical guidelines. Mice were randomized into groups of 4 mice according to body weight. On day 0 of the study, animals received a single subcutaneous dose of 1 mg / kg or 5 mg / kg siRNA dissolved in phosphate buffered saline (PBS) or PBS alone as a control. Mice viability, body weight and behavior were monitored during the study without pathological views. Serum samples were taken on days 4, 10 and 14 prior to application. On day 14, the study was terminated, animals were euthanized, liver samples were flash frozen and stored at -80 ° C for further analysis. For analysis, Stratec's InviTrap Spin Tissue RNA Mini Kit was used to isolate RNA according to the manufacturer's protocol. QPCRs were performed in a single-plex 384-well format on Applied Biosystems QuantStudio 6 devices using C3 and actin-specific primer probe sets and Takayon ™ One-Step Low RoxProbe 5X MasterMix dTTP. Differences in expression were calculated using the delta-delta CT method and the relative expression of C3 to the housekeeping gene actin normalized to PBS control experiments was used for comparison of various siRNAs. EV0107, EV0313 and EV0110 induced dose-dependent knockdown of liver C3 mRNA. Maximum achieved knockdown was observed using siRNA EV0107 (57%) and EV0110 (61%) with 5 mg / kg siRNA, respectively. The results are shown in Figure 6A. The figure shows the relative expression of C3 mRNA in% in mouse liver 14 days after a single dose of GalNAc conjugated siRNA EV0107, EV0313 and EV0110. The single-stranded identities that form each siRNA duplex and their sequences can be found in the table at the end of the description. The data are shown in the bar graph as mean ± SD (n = 4 per group).

Serum samples were analyzed using a commercially available C3 ELISA kit. Analysis was performed according to the manufacturer's protocol and C3 serum levels were calculated for each pre-dose level. The results are shown in Figure 6B. The figure shows the 4th, 10th, and 14th days of the study before the administration of 5 mg / kg EV0313, EV0110 and EV0107 GalNAc conjugated siRNA, and after the administration of 5 mg / kg EV0313, EV0110 and EV0107 GalNAc conjugated siRNA. Shows relative C3 protein serum levels in% from mouse serum samples collected in. Data are shown as mean ± SD (n = 3 or 4 per group).

(Example 10)
In vitro studies in primary cultured mouse, human and cynomolgus monkey hepatocytes demonstrating the C3 knockdown efficacy of siRNA-GalNAc conjugates tested at 1nM, 10nM and 100nM.
Expression of C3 mRNA after incubation with GalNAc siRNA conjugates EV0201, EV0203, EV0204, EV0205 and EV0207 at 1 nM, 10 nM and 100 nM was measured (Fig. 7). The siRNA sequences and modifications are listed in Table 3 (Table 4) and Table 5 (Table 6). The mRNA levels of the mouse-keeping gene actin served as a housekeeping control. Human and cynomolgus monkey primary cultured hepatocytes were seeded on a collagen I-coated 96-well plate (Life Technologies) at a density of 40,000 cells per well. Mouse hepatocytes were seeded at a density of 25,000 cells per well. GalNAc conjugated siRNA was added to final siRNA concentrations of 100 nM, 10 nM and 1 nM immediately after sowing in a pre-defined medium. The plates were then incubated at 37 ° C. in a 5% CO ₂ atmosphere for 24 hours. Subsequently, cells were lysed and RNA was isolated using the InviTrap RNA Cell HTS96 Kit / C (Stratec).

Gene expression analysis of 10 μl RNA solution by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) performed using amplicon sets / sequences for ACTB (Eurogentec) and C3 (BioTez, Berlin, Germany), respectively. Used for. RT-qPCR reactions were performed using ABI StepOne Plus (Applied Biosystems, part of Thermo Fisher Scientific, USA, Massachusetts) and standard protocols for RT-PCR (48 ° C 30 minutes, 95 ° C 10 minutes, Performed using 40 cycles of 15 seconds at 95 ° C, then 1 minute at 60 ° C). Data were calculated by using the comparative CT method, which is also known as the 2-delta delta Ct method. siRNA EV0201, EV0203, EV0204, EV0205 and EV0207 show dose-dependent inhibition of C3 mRNA in primary cultured hepatocytes.

(Example 11)
In vivo studies showing knockdown of C3 mRNA and serum proteins in mouse liver tissue after a single subcutaneous dose of GalNAc-conjugated siRNA up to 10 mg / kg.
The siRNA sequences and modifications are listed in Table 3 (Table 4) and Table 5 (Table 6). The mRNA levels of the mouse-keeping gene actin served as a housekeeping control. Male C57BL / 6N mice approximately 8 weeks old were obtained from CHARLES RIVER, Sulzfeld, Germany. Animal studies were conducted in compliance with the Hungarian Act 1998: XXVIII Principles (latest amendments by Act 2011 CLVIII), which regulate animal protection, and in Government Decree 40/2013 on Animal Care. Mice were assigned to a group of 4 mice. On day 0 of the study, animals received a single subcutaneous dose of 5 mg / kg or 10 mg / kg siRNA dissolved in phosphate buffered saline (PBS) or PBS alone as a control. Mice viability, body weight and behavior were monitored during the study without pathological views. Serum samples were taken on days 4, 10, 14, 21, 28, 35 and 42 before application. On days 14 and 42, half of the groups were terminated, animals were euthanized, liver samples were flash frozen and stored at -80 ° C for further analysis.

For analysis, Stratec's InviTrap Spin Tissue RNA Mini Kit was used to isolate RNA according to the manufacturer's protocol. RT-qPCR was performed in a single-plex 384-well format on Applied Biosystems QuantStudio6 devices using C3 and actin-specific primer probe sets and Takayon ™ One-Step Low RoxProbe 5X MasterMix dTTP. Differences in expression were calculated using the delta-delta CT method and the relative expression of C3 to the housekeeping gene actin normalized to the PBS group was used for comparison of various siRNAs.

All siRNAs tested (EV0201, EV0203, EV0204, EV0205 and EV0207) inhibit C3 mRNA expression by more than 70% 14 days after a single dose of 5 mg / kg or 10 mg / kg (Fig. 8A). .. After 42 days, inhibition of C3 expression by EV0203, EV0204, EV0205 and EV0207 was still more than 80% knockdown at a siRNA dose of 10 mg / kg (Fig. 8B).

For C3 protein level analysis, serum samples were measured using a commercially available C3 ELISA kit. The analysis was performed according to the manufacturer's protocol and C3 serum level% was calculated against the baseline / pre-application group mean and against the time-matched PBS control mean. The data for C3 protein analysis are very similar to the results from RNA analysis (Figure 9). EV0203, EV0204, EV0205 and EV0207 were able to induce sustained C3 serum depletion. Up to 80% reduction was obtained for EV0203 after 42 days of single application of 10 mg / kg (Fig. 9B). Further doses of EV0203 were tested in a 28-day experiment (Fig. 9F). A surprisingly low dose of 3 mg / kg EV0203 was able to reduce serum C3 levels by more than 50% from day 7 to at least day 28.

(Example 12)
EV0203 will be tested in a mouse disease model of C3 glomerulosis. Mice used as a model for C3 glomerular disease are complement H factor (Cfh) deficient mice or heterozygous mice with respect to Cfh. Animals are treated once or multiple times with various doses of EV0203. Kidney, liver and serum levels of complement factors, including C3, are analyzed different times after subcutaneous siRNA application. After treatment with EV0203, C3 protein levels are predicted to be reduced in serum and C3 mRNA levels are predicted to be reduced in liver tissue. Treatment is expected to result in a reduction in complement deposition (ie, C3 deposition) in at least some parts of the kidney. Fragmentation of complement B factor and / or C3 is also expected to be reduced. These reductions in fragmentation are expected to accompany reduced activation of alternative pathways. Treatment with EV0203 is also expected to reduce proteinuria and hematuria, two important symptoms of C3 glomerulopathy. Therefore, EV0203 is expected to be a potent treatment for C3-related diseases, especially for C3 glomerulopathy and / or at least its symptoms.

Description The following description represents aspects of the invention.

1. A double-stranded nucleic acid for inhibiting the expression of complement component C3, wherein the nucleic acid contains a first strand and a second strand, and the sequence of the first strand is SEQ ID NO: 361, 95, 111, 125, 131, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, Any one of 93, 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 127, 129 or 133 and at least 15 different nucleotides of 3 or less. A nucleic acid containing a sequence of nucleotides.

2. (a) The sequence of the first strand contains any one of the sequences of the first strand in Table 1 and a sequence of at least 18 nucleotides different from 3 or less nucleotides, and is optional. In the selection, the sequence of the second strand contains the sequence of the second strand in the same row of the table and the sequence of at least 18 nucleotides with no more than 3 nucleotides different.
(b) The sequence of the first strand contains any one of the sequences of the first strand in Table 1 and a sequence of at least 18 nucleotides with one or less different nucleotides, optionally. , The sequence of the second strand contains a sequence of at least 18 nucleotides with one or less different nucleotides from the sequence of the second strand in the same row of the table.
(c) The sequence of the first strand contains the sequence of at least 18 nucleotides of any one of the sequences of the first strand in Table 1 and optionally the sequence of the second strand , Containing a sequence of at least 18 nucleotides in the second strand sequence of the same row in the table, or
(d) The sequence of the first strand consists of any one of the sequences of the first strand in Table 1 and, optionally, the sequence of the second strand is the first in the same row of the table. It consists of an array of two strands.
Table 1 shows:

The nucleic acid according to description 1.

3. The sequence of the first strand contains the sequence of SEQ ID NO: 361 and optionally the sequence of the second strand contains the sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 112 or the first. The sequence of strands contains the sequence of SEQ ID NO: 95 and, optionally, the sequence of the second strand contains the sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 96, or the sequence of the first strand. However, the sequence of SEQ ID NO: 125 is included, and optionally, the sequence of the second strand contains the sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 126, or the sequence of the first strand is SEQ ID NO: The nucleic acid according to any one of the above descriptions, comprising 131 sequences and optionally the sequence of the second strand comprising the sequence of at least 15 nucleotides of the sequence of SEQ ID NO: 132.

4. A double-stranded nucleic acid capable of inhibiting the expression of complement component C3 for pharmaceutical use.

5. The nucleic acid according to any one of the above statements, wherein the first and second strands are separate strands, each 18 to 25 nucleotides in length.

6. The nucleic acid according to any one of the above descriptions, wherein the first and second strands form a double-stranded region 17 to 25 nucleotides in length.

7. The nucleic acid according to any one of the above descriptions, wherein the double-stranded region consists of 17 to 25 consecutive nucleotide base pairs.

8.a) Is it a blunt end at both ends?
b) Have an overhang at one end and a blunt end at the other end, or
c) have overhangs at both ends,
The nucleic acid according to any one of the above descriptions.

9. The nucleic acid according to any one of the above descriptions, which is siRNA.

10. Nucleic acid according to any one of the above statements that mediates RNA interference.

11. The nucleic acid according to any one of the above statements, wherein at least one nucleotide in the first and / or second strand is a modified nucleotide.

12. At least nucleotides 2 and 14 of the first strand are modified by the first modification and the nucleotides are numbered sequentially starting with nucleotide number 1 at the 5'end of the first strand. The nucleic acid according to any one of the above descriptions.

13. Each even numbered nucleotide in the first strand is modified by the first modification, and the nucleotides are numbered sequentially starting with nucleotide number 1 at the 5'end of the first strand. The nucleic acid according to any one of the above-mentioned descriptions.

14. The nucleic acid of description 12 or 13, wherein the odd numbered nucleotides of the first strand are modified by the second modification, the second modification being different from the first modification.

15. Description 12: The nucleotide of the second strand at the position corresponding to the even numbered nucleotide of the first strand is modified by the third modification, the third modification being different from the first modification. Nucleic acid according to to 14.

16. When the nucleotide of the second strand at the position corresponding to the nucleotide numbered to the odd number of the first strand is modified by the fourth modification and the second modification and / or the third modification is present. , The nucleic acid according to description 12-15, wherein the fourth modification is different from the second modification and also different from the third modification.

17. Nucleotides 11 or 13 of the first strand or nucleotides 11 and 13 or nucleotides of the second strand (s) at positions corresponding to nucleotides 11-13 are modified by the fourth modification, preferably. , The nucleic acid of description 12-14, wherein the nucleotide of the second strand, which is not modified by the fourth modification, is modified by the third modification.

18. If both the first and fourth modifications are present in the nucleic acid, the first modification is the same as the fourth modification, preferably both the second and third modifications. The nucleic acid of description 12-17, wherein the second modification is the same as the third modification if is present in the nucleic acid.

19. If the first modification is a 2'-F modification and the second modification is present in the nucleic acid, preferably the 2'-OMe modification and the third modification is present in the nucleic acid. Descriptions 12-18, wherein the nucleic acid is preferably a 2'-OMe modification, preferably a 2'-F modification if the fourth modification is present in the nucleic acid.

20. The nucleic acid according to any one of the above statements, wherein each of the first and second strand nucleotides is a modified nucleotide.

21. The first strand has a terminal 5'(E) -vinylphosphonate nucleotide at its 5'end, and the terminal 5'(E) -vinylphosphonate nucleotide is preferably the second in the first strand. The nucleic acid according to any one of the above descriptions, which is linked to a nucleotide by a phosphodiester bond.

22. The nucleic acid comprises a phosphodiester bond between the 2 or 3 3'nucleotides and / or 5'nucleotides at the end of the 1st and / or 2nd strand, preferably the binding between the remaining nucleotides. The nucleic acid according to any one of the above descriptions, which is a phosphodiester bond.

23. Contains a phosphorodithioate bond between each of the 2, 3 or 4 terminal nucleotides at the 3'end of the first strand, and / or 2 at the 3'end of the second strand. A phosphorodithioate bond between each of the 3, 3 or 4 terminal nucleotides, and / or between the 2, 3 or 4 terminal nucleotides at the 5'end of the second strand, respectively. 1. Nucleic acid described in one.

24. If no phosphorodithioate bond is present at its end, between each of the three terminal 3'nucleotides on the first strand and / or between each of the three terminal 5'nucleotides, and /. Or the nucleic acid of description 23, comprising a phosphorothioate bond between each of the three terminal 3'nucleotides and / or between each of the three terminal 5'nucleotides on the second strand.

25. Nucleic acids in both strands except the bond between the two terminal nucleotides at the 3'end of the first strand and the bond between the two terminal nucleotides at the 3'and 5'ends of the second strand. The nucleic acid according to description 23, wherein all the bonds between them are phosphodiester bonds.

26. The nucleic acid according to any one of the above descriptions, conjugated to a ligand.

27. The ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, the linker conjugates at least one GalNAc moiety or derivative thereof to the nucleic acid. The nucleic acid according to description 26.

28. Equation (II):
[SX ¹ -PX ² ] ₃ -AX ^3- (II)
(During the ceremony,
S represents a saccharide, preferably the saccharide is N-acetylgalactosamine.
X ¹ represents C ₃ to C ₆ alkylene or (-CH ₂ -CH ₂ -O) _m (-CH ₂ ) _2- (in the formula, m is 1, 2 or 3).
P is phosphate or modified phosphate, preferably thiophosphate, and
X ² is an alkylene or an alkylene ether of the formula (-CH ₂ ) _n -O-CH _2- (in the formula, n = 1 to 6).
A is a branch unit,
X ³ represents the crosslinkable unit
Here, the nucleic acid as defined in any of description 1-27 is conjugated to X ³ by phosphate or modified phosphate, preferably thiophosphate).
The nucleic acid according to any one of descriptions 1-27, which is conjugated to a ligand containing the compound of.

29. The first strand of nucleic acid is in formula (V):

(In the formula, b is 0 or 1)
The second chain is the compound of formula (VI):

(During the ceremony,
c and d are independently 0 or 1 and
Z ₁ and Z ₂ are the first and second strands of nucleic acid, respectively.
Y is independently O or S,
n is independently O, 1, 2 or 3,
L ₁ is a ligand-bound linker, where L ₁ is the same or different in equations (V) and (VI), and L ₁ is more than one in the same equation. If many are present, they are the same or different in equations (V) and (VI).
b + c + d is 2 or 3)
The nucleic acid according to any one of descriptions 1-27, which is a compound of.

30. Containing nucleic acids and delivery vehicles and / or physiologically acceptable excipients and / or carriers and / or diluents and / or buffers and / or preservatives according to any one of the preceding statements. Composition.

31. A composition comprising the nucleic acid according to any one of Descriptions 1-29 and a further therapeutic agent selected from the group comprising oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies and peptides.

32. The nucleic acid according to any one of Descriptions 1-3 or 5 to 29 for use as a pharmaceutical or the composition according to Description 30 or 31.

33. Nuclei or Descriptions 30 described in any one of Descriptions 1-29 for the prevention of a disease, disorder or syndrome, reduction of the risk of suffering from a disease, disorder or syndrome, or use in the treatment of a disease, disorder or syndrome. The composition according to any one of 32.

34. The nucleic acid or composition according to description 33, wherein the disease, disorder or syndrome is a complement-mediated disease, disorder or syndrome.

35. Diseases, disorders or syndromes are associated with aberrant and / or overactivation of the complement pathway and / or overexpression or ectopic expression or localization or accumulation of C3, in description 33 or 34. The nucleic acid or composition of the description.

36. Diseases, disorders or syndromes
a) C3 glomerulonephritis (C3G), paroxysmal nocturnal hemoglobinuria (PNH), atypical lupus erythematosus syndrome (aHUS), lupus nephritis, IgA nephropathy (IgA N), primary membranous nephropathy, immunity Complex-mediated glomerulonephritis (IC-mediated GN), post-infection glomerulonephritis (PIGN), systemic lupus erythematosus (SLE), ischemia-reperfusion injury, age-related luteal degeneration (AMD), rheumatoid arthritis (RA), Whether selected from the group including antineupus erythematosus autoantibody-related vasculitis (ANCA-AV), disbiosis periodontal disease, malaria anemia and septicemia
b) Includes C3 glomerulopathy (C3G), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic urotoxicity syndrome (aHUS), lupus nephritis, IgA nephropathy (IgA N) and primary membranous nephropathy Selected from the group or
c) C3 glomerulopathy (C3G)
The nucleic acid or composition according to any one of the descriptions 33 to 35.

37. Prevention of a disease, disorder or syndrome, reduction of the risk of suffering from a disease, disorder or syndrome, or any of the nucleic acids described in any one of Descriptions 1-29 or 30-31 of Descriptions in the treatment of a disease, disorder or syndrome. Use of the composition according to one of the above, wherein the disease, disorder or syndrome is preferably C3 glomerular disease (C3G).

38. A pharmaceutically effective dose of the nucleic acid according to any one of Descriptions 1-29 or 32-36 or the composition according to any one of Descriptions 30-36 requires treatment. A method of preventing a disease, disorder or syndrome, reducing the risk of developing the disease, disorder or syndrome, or treating the disease, disorder or syndrome, including the step of administering to an individual, preferably nucleic acid or A method in which the composition is administered to a subject subcutaneously, intravenously, or by oral, rectal, or intraperitoneal administration.

Summary table

Abbreviations as shown in the table of abbreviations above may be used herein. The list of abbreviations may not be exhaustive and further abbreviations and their meanings may be found throughout this document.

Claims (15)

A double-stranded nucleic acid for inhibiting the expression of complement component C3, wherein the nucleic acid comprises a first strand and a second strand, and the sequence of the first strand is SEQ ID NO: 361, 95, 111, 125, 131, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, Any one of 97, 99, 101, 103, 105, 107, 109, 113, 115, 117, 119, 121, 123, 127, 129 or 133 and at least 15 nucleotides with 3 or less nucleotides different. Nucleic acid, including sequences. A double-stranded nucleic acid capable of inhibiting the expression of complement component C3 for pharmaceutical use. The nucleic acid according to claim 1 or 2, wherein the first strand and the second strand form a double-stranded region having a length of 17 nucleotides to 25 nucleotides. The nucleic acid according to any one of claims 1 to 3, which mediates RNA interference. Any one of claims 1 to 4, wherein at least one nucleotide in the first and / or second strand is a modified nucleotide, preferably a non-naturally occurring nucleotide such as a 2'-F modified nucleotide. Nucleic acid according to. At least nucleotides 2 and 14 of the first strand are modified by the first modification and the nucleotides are numbered sequentially starting with nucleotide number 1 at the 5'end of the first strand. , The nucleic acid according to any one of claims 1 to 5. The nucleic acid according to any one of claims 1 to 6, wherein the first strand has a terminal 5'(E) -vinylphosphonate nucleotide at its 5'end. The nucleic acid comprises a phosphodiester bond between the two or three 3'nucleotides and / or 5'nucleotides at the end of the first and / or the second strand, preferably the binding between the remaining nucleotides. , The nucleic acid according to any one of claims 1 to 7, which is a phosphodiester bond. Containing a phosphorodithioate bond between each of the 2, 3 or 4 terminal nucleotides at the 3'end of the first strand, and / or 2 at the 3'end of the second strand, A phosphorodithioate bond between each of the 3 or 4 terminal nucleotides and / or a phospho between the 2 or 3 or 4 terminal nucleotides at the 5'end of the second strand. Any of claims 1-8, comprising a logithioate bond and comprising a bond other than a phosphorodithioate bond between two, three or four terminal nucleotides at the 5'end of the first strand. The nucleic acid according to one item. The nucleic acid according to any one of claims 1 to 9, which is conjugated to a ligand. The ligand comprises (i) one or more N-acetylgalactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker brings at least one of the GalNAc moieties or derivatives thereof to the nucleic acid. The nucleic acid according to claim 10, which is conjugated. The nucleic acid and delivery vehicle and / or physiologically acceptable excipient and / or carrier and / or diluent and / or buffer and / or preservative according to any one of claims 1 to 11. / Or a composition comprising an additional therapeutic agent selected from the group comprising oligonucleotides, small molecules, monoclonal antibodies, polyclonal antibodies and peptides. The nucleic acid according to any one of claims 1 and 3 to 11 or the composition according to claim 12. The nucleic acid or claim according to any one of claims 1 and 3 to 11 for prevention of a disease, disorder or syndrome, reduction of the risk of suffering from a disease, disorder or syndrome, or use in the treatment of a disease, disorder or syndrome. Item 12. A nucleic acid or composition according to Item 12, wherein the disease, disorder or syndrome is preferably a complement-mediated disease, disorder or syndrome. The nucleic acid or claim 12 according to any one of claims 1 and 3 to 11 in the prevention of a disease, disorder or syndrome, reduction of the risk of suffering from a disease, disorder or syndrome, or the treatment of a disease, disorder or syndrome. The use of the composition of, wherein the disease, disorder or syndrome is preferably C3 glomerulopathy (C3G).

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